Multimeric coding nucleic acid and uses thereof

ABSTRACT

The present invention provides, among other things, multimeric coding nucleic acids that exhibit superior stability for in vivo and in vitro use. In some embodiments, a multimeric coding nucleic acid (MCNA) comprises two or more encoding polynucleotides linked via 3′ ends such that the multimeric coding nucleic acid compound comprises two or more 5′ ends.

RELATED APPLICATIONS

This application is a divisional Application of U.S. Non-Provisional application Ser. No. 15/482,431, filed Apr. 7, 2017, which claims priority to U.S. Provisional Application Ser. No. 62/320,073, filed Apr. 8, 2016, the disclosure of which is hereby incorporated by reference.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing (submitted electronically as a .txt file named “SL_SHR-1237US” on Apr. 7, 2017. The .txt file was generated Apr. 7, 2017 and is 49,249 bytes in size. The entire contents of the Sequence Listing are herein incorporated by reference.

BACKGROUND

Nucleic acid-based technologies are increasingly important for various therapeutic applications including, but not limited to, messenger RNA therapy, gene therapy, and gene editing, to name but a few. Such therapeutic applications typically require administration of exogenous polynucleotides (e.g. DNA or RNA), which is often hampered by the limited stability of such polynucleotides. For example, following their administration to a subject, many polynucleotides may be subject to nuclease (e.g. exonuclease and/or endonuclease) degradation. Nuclease degradation may negatively influence the capability of a polynucleotide to reach a target cell or to be transcribed and/or translated, the result of which is to preclude the exogenous polynucleotide from exerting an intended therapeutic effect.

SUMMARY OF THE INVENTION

The present invention provides, among other things, multimeric coding nucleic acids that exhibit superior stability for in vivo and in vitro use. The present invention also permits increased complexity and efficiency for nucleic acid based therapeutics.

In some aspects, the present invention provides a multimeric coding nucleic acid (MCNA) comprising one or more coding polynucleotides linked to one or more non-coding polynucleotides via a 3′ end linkage between two or more of the polynucleotides (coding or non-coding) such that the MCNA compound comprises two or more 5′ ends. In some embodiments, one or more of the 5′ ends is modified to include a 5′ end cap structure. In certain embodiments, one or more of the coding polynucleotides having a 5′ end comprises a 5′ end cap structure to facilitate translation of the coding polynucleotides. In certain embodiments, one or more of the polynucleotides having a 5′ end structure comprises a 5′ end cap structure to facilitate stability of the MCNA.

In some aspects, the present invention provides a multimeric coding nucleic acid (MCNA) comprising two or more encoding polynucleotides linked via 3′ ends such that the multimeric coding nucleic acid compound comprises two or more 5′ ends. In some embodiments, each of the two or more encoding polynucleotides is a synthetic polyribonucleotide. In some embodiments, each of the two or more encoding polynucleotides is a synthetic polydeoxyribonucleotide. In some embodiments, each of the two or more encoding polynucleotides is a synthetic polydeoxyribonucleotide or a polyribonucleotide. In some embodiments, each of the two or more encoding polynucleotides encodes a protein of interest. In some embodiments, each of the two or more encoding polynucleotides encodes a same protein. In some embodiments, each of the two or more encoding polynucleotides encodes a distinct protein.

In some embodiments, the MCNA compound comprises three or more encoding polynucleotides. In some embodiments, the compound comprises four or more encoding polynucleotides. In some embodiments, the compound comprises five or more encoding polynucleotides.

In some embodiments, one or more of the encoding polynucleotides comprise a 5′ untranslated region (5′ UTR) and/or a 3′ untranslated region (3′ UTR). In some embodiments, the one or more of the encoding polynucleotides comprise a 3′ UTR. In some embodiments, the 3′ UTR is 5-2,000 nucleotides in length. In some embodiments, the 3′ UTR comprises a plurality of multi-A segments with spacers in between. In some embodiments, each of the multi-A segments comprises 8-50 consecutive adenosines. In some embodiments, the plurality of multi-A segments range from 1-100. In some embodiments, the spacers are of varying lengths ranging from 5-100. In some embodiments, the spacers comprise DNA, RNA and/or modified bases. In some embodiments, the modified bases are selected from 2′-OMe-A, 2′-OMe-G, 2′-OMe-C, 2′-OMe-U, 2′-F-A, 2′-F-G, 2′-F-C, 2′-F-U, LNA-A, LNA-G, LNA-C, LNA-U, N6-methyl-adenosine, 2-thiouridine (2sU), 5-methyl-cytidine (5mC), pseudouridine (ΨU), and 1-methyl-pseudouridine. In some embodiments, the 3′ UTR comprises a pseudoknot structure. In some embodiments, the 3′ UTR is not followed with a polyadenylation (poly-A) tail. In some embodiments, one or more of the encoding polynucleotides comprise a poly-A tail. In some embodiments, the poly-A tail is 25-5,000 nucleotides in length. In some embodiments, the 3′ UTR binds to poly-A binding proteins (PABPs). In some embodiments, the 3′ UTR comprises a “kissing loop” sequence motif.

In some embodiments, the 3′ ends of the two or more encoding polynucleotides are linked via an oligonucleotide bridge comprising a 3′-3′ inverted phosphodiester linkage. In some embodiments, the nucleotides comprising the oligonucleotide bridge are selected from the group consisting of 2′-OMe-A, 2′-OMe-G, 2′-OMe-C, 2′-OMe-U, 2′-F-A, 2′-F-G, 2′-F-C, 2′-F-U, LNA-A, LNA-G, LNA-C, LNA-U, N6-methyl-adenosine, 2-thiouridine (2sU), 5-methyl-cytidine (5mC), pseudouridine (ψU), and 1-methyl-pseudouridine. In some embodiments, the oligonucleotide bridge comprises at least one covalent link to an active moiety. In some embodiments, the active moiety is a targeting group, peptide, contrast agent, small molecule, protein, DNA and/or RNA. In some embodiments, nucleotides proximal to the 3′-3′ inverted linkage are functionalized with one or more tri-antennary GalNac targeting agents.

In some embodiments, the encoding polynucleotides comprise one or more modified nucleotides. In some embodiments, the modified nucleotides are selected from the group consisting of 2′-OMe-A, 2′-OMe-G, 2′-OMe-C, 2′-OMe-U, 2′-F-A, 2′-F-G, 2′-F-C, 2′-F-U, LNA-A, LNA-G, LNA-C, LNA-U, N6-methyl-adenosine, 2-thiouridine (2sU), 5-methyl-cytidine (5mC), pseudouridine (ψU), and 1-methyl-pseudouridine. In some embodiments, the modified nucleotides substitute 1-100% of corresponding native bases. In some embodiments, the at least 25% of uridines are replaced with 2-thiouridines. In some embodiments, 100% of cytidines are replaced with 5-methylcytidines. In some embodiments, the modified nucleotides are further modified with a 4′-thio substitution on the ribose ring. In some embodiments, the native nucleotides are modified with a 4′-thio substitution on the ribose ring.

In some embodiments, one or more encoding polynucleotides in the MCNA comprise a polynucleotide portion that encodes a therapeutic protein. In some embodiments, one or more encoding polynucleotides in the MCNA comprise a polynucleotide portion that encodes an enzyme, a receptor, a ligand, a light chain or heavy chain of an antibody, a nuclease, or a DNA-binding protein. In certain embodiments, one or more encoding polynucleotides in the MCNA comprise a polynucleotide portion that encodes a nuclease.

In some embodiments, the two or more encoding polynucleotides in the MCNA each comprise a polynucleotide portion that encodes a therapeutic protein. In some embodiments, the two or more encoding polynucleotides in the MCNA each comprise a polynucleotide portion that encodes an enzyme, a receptor, a ligand, a light chain or heavy chain of an antibody, a nuclease, and/or a DNA-binding protein. In some embodiments, the two or more encoding polynucleotides in the MCNA each comprise a polynucleotide portion that encodes a nuclease.

In some embodiments, a first encoding polynucleotide in the MCNA comprises a polynucleotide portion that encodes a first protein and a second encoding polynucleotide in the MCNA comprising a polynucleotide portion that encodes a second protein that is the same protein as the first protein. In some embodiments, a first encoding polynucleotide in the MCNA comprises a polynucleotide portion that encodes a first protein and a second encoding polynucleotide in the MCNA comprises a polynucleotide portion that encodes a second protein that is distinct from the first protein. In certain embodiments, a first encoding polynucleotide in the MCNA comprises a polynucleotide portion that encodes a first protein in a class of an enzyme, a receptor, a ligand, a light chain or heavy chain of an antibody, a nuclease, or a DNA-binding protein, and a second encoding polynucleotide in the MCNA comprises a polynucleotide portion that encodes a second protein that is distinct from the first protein but in the same class as the first protein. In certain embodiments, a first encoding polynucleotide in the MCNA comprises a polynucleotide portion that encodes a first protein in a class of an enzyme, a receptor, a ligand, a light chain or heavy chain of an antibody, a nuclease, or a DNA-binding protein, and a second encoding polynucleotide in the MCNA comprises a polynucleotide portion that encodes a second protein that is distinct from the first protein and in a different class from the first protein. In certain embodiments, a first encoding polynucleotide in the MCNA comprises a polynucleotide portion that encodes a light chain of an antibody and a second encoding polynucleotide in the MCNA comprises a polynucleotide portion that encodes a heavy chain in the antibody.

In some aspects, the present invention provides a multimeric nucleic acid (MNA) comprising two or more polynucleotides linked via at least one 3′ end linkage between two or more of the polynucleotides such that the MNA compound comprises two or more 5′ ends. In some embodiments, one or more of the 5′ ends is modified to facilitate stability of the MNA. In certain embodiments, the two or more polynucleotides linked via the at least one 3′ end linkage each are non-coding nucleotides.

In some aspects, the present invention provides a composition comprising the MCNA as described above, encapsulated or complexed with a delivery vehicle. In some embodiments, the delivery vehicle is selected from the group consisting of liposomes, lipid nanoparticles, solid-lipid nanoparticles, polymers, viruses, sol-gels, and nanogels.

In some aspects, the present invention provides methods of delivering MCNA for in vivo protein production, comprising administering the MCNA as described above to a subject in need of delivery. In some embodiments, the MCNA is administered via a route of delivery selected from the group consisting of intravenous delivery, subcutaneous delivery, oral delivery, subdermal delivery, ocular delivery, intratracheal injection pulmonary delivery (e.g. nebulization), intramuscular delivery, intrathecal delivery, or intraarticular delivery.

It is to be understood that all embodiments as described above are applicable to all aspects of the present invention.

BRIEF DESCRIPTION OF THE DRAWING

The drawings are for illustration purposes only, not for limitation.

FIG. 1 shows an exemplary MCNA comprising two RNA species linked via a 3′-3′ inverted RNA nucleotide dimer.

FIG. 2 shows an exemplary MCNA comprising two RNA species linked via a 3′-3′ inverted RNA nucleotide dimer wherein the MCNA is functionalized with a tri-antennary GalNac targeting agent.

FIG. 3 shows an exemplary MCNA comprising two RNA species linked via a 3′-3′ inverted RNA nucleotide dimer wherein the MCNA is functionalized with two tri-antennary GalNac targeting agent.

FIG. 4 shows a general scheme for synthesis of MCNA.

FIG. 5 shows exemplary results of synthesized EPO MCNA detected via gel electrophoresis. Constructs were synthesized under the following conditions: RNA Ligase 1 (A); RNA Ligase 1+10% PEG (B); and RNA Ligase 2 (C).

FIG. 6 shows exemplary results of synthesized EPO MCNA detected via gel electrophoresis. Lane 1 show capped EPO RNA with no tail. Lane 2 shows an EPO MCNA mixture with no DNAse treatment. Lane 3 shows an EPO MCNA mixture treated with DNAse.

FIG. 7 shows an exemplary graph of the level of hEPO protein secreted after transfection of HEK293T cells with synthetic constructs comprising untailed EPO mRNA or MCNA comprising hEPO mRNA (1 microgram per construct).

FIG. 8 shows exemplary results of synthesized EPO MCNA detected via gel electrophoresis. Lane 1 contains an RNA Ladder, Lane 2 contains a ligation product for EPO MCNA that was not purified, Lane 3 contains purified unreacted/partially reacted product and Lane 4 contains purified EPO MCNA ligation product.

FIG. 9 shows an exemplary graph of the level of hEPO protein secreted after transfection of HEK293T cells with synthetic constructs comprising untailed EPO mRNA or purified MCNA comprising hEPO mRNA (250 nanogram per construct).

FIG. 10 shows an exemplary graph of the level of hOTC protein activity measured in cell lysate after transfection of HEK293T cells with synthetic constructs comprising untailed hOTC mRNA (hOTC monomer) or MCNA comprising hOTC mRNA.

FIG. 11 shows an exemplary graph of the level of hPAH protein produced after transfection of HEK293T cells with synthetic constructs comprising untailed hPAH mRNA (hPAH monomer) or MCNA comprising hPAH mRNA.

FIG. 12 shows an exemplary Western blot demonstrating hCFTR protein production after transfection of HEK293T cells with synthetic constructs comprising untailed hCFTR mRNA (hCFTR monomer) or MCNA comprising hCFTR mRNA.

FIG. 13 shows an exemplary graph of citrulline production measured in livers of mice after treatment with hOTC MCNA encapsulated in lipid nanoparticles.

FIG. 14 shows an exemplary Western blot demonstrating hOTC production detected in livers of mice after treatment with hOTC MCNA or hOTC monomers encapsulated in lipid nanoparticles.

FIG. 15 shows an exemplary graph of citrulline production measured in livers of mice after treatment with hOTC mRNA encapsulated in lipid nanoparticles.

FIG. 16 shows an exemplary graph comparing citrulline production 1 week after administration as a percentage of citrulline production 24 hours after administration in mice treated with hOTC mRNA or hOTC MCNA encapsulated in lipid nanoparticles.

FIG. 17 shows an exemplary graph of hPAH protein detected in livers of PAH knock-out (KO) mice 24 hours after they were administered either hPAH MCNA or hPAH monomers encapsulated in lipid nanoparticles.

FIG. 18 shows an exemplary graph of serum phenylalanine levels in PAH knock-out (KO) mice 24 hours after they were administered either hPAH MCNA or hPAH monomers encapsulated in lipid nanoparticles.

FIG. 19 shows an exemplary graph of hEPO protein detected in the serum of wild-type mice 24 hours after they were administered either hEPO MCNA or hEPO monomers encapsulated in lipid nanoparticles.

FIG. 20 shows exemplary immunohistochemical detection of human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR) protein in CFTR KO mouse lung 24 hours and 7 days after treatment with hCFTR MCNA encapsulated in lipid nanoparticles via aerosolization.

DEFINITIONS

In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

Amino acid: As used herein, the term “amino acid,” in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an 1-amino acid. “Standard amino acid” refers to any of the twenty standard 1-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, “synthetic amino acid” encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond. Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term “amino acid” is used interchangeably with “amino acid residue,” and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Biologically active: As used herein, the term “biologically active” refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.

Delivery: As used herein, the term “delivery” encompasses both local and systemic delivery. For example, delivery of MCNA encompasses situations in which an MCNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an MCNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery).

Expression: As used herein, “expression” of a nucleic acid sequence refers to translation of an MCNA into a polypeptide, assemble multiple polypeptides into an intact protein (e.g., enzyme) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., enzyme). In this application, the terms “expression” and “production,” and grammatical equivalent, are used inter-changeably.

Functional: As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

Half-life: As used herein, the term “half-life” is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.

Improve, increase, or reduce: As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.

In Vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

In Vivo: As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).

messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” or “mRNA” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5′ to 3′ direction unless otherwise indicated. A typical mRNA molecule has a 5′ end and a 3′ end. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

Nucleic acid: As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA and/or cDNA.

Patient: As used herein, the term “patient” or “subject” refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. A human includes pre- and post-natal forms.

Pharmaceutically acceptable: The term “pharmaceutically acceptable”, as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable salt: Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or rnalonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium. quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.

Systemic distribution or delivery: As used herein, the terms “systemic distribution,” “systemic delivery,” or grammatical equivalent, refer to a delivery or distribution mechanism or approach that affect the entire body or an entire organism. Typically, systemic distribution or delivery is accomplished via body's circulation system, e.g., blood stream. Compared to the definition of “local distribution or delivery.”

Subject: As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

Target tissues: As used herein, the term “target tissues” refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease-associated pathology, symptom, or feature.

Therapeutically effective amount: As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

Treating: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

DETAILED DESCRIPTION

The present invention provides, among other things, methods for synthesizing and compositions comprising multimeric coding nucleic acids (MCNA). In particular, the present invention provides MCNA compounds comprising two or more encoding polynucleotides linked via their 3′ ends such that the MCNA compound comprises two or more 5′ ends and methods of synthesizing the same. In some embodiments, each of the two or more encoding polynucleotides is a synthetic polyribonucleotide. In some embodiments, each of the two or more encoding polynucleotides is a synthetic polydeoxyribonucleotide. In some embodiments, a synthetic polyribonucleotide or polydeoxyribonucleotide of the invention codes for a polypeptide, protein, enzyme, antibody, or receptor. In some embodiments, the present invention provides a multimeric nucleic acid (MNA) comprising two or more polynucleotides linked via at least one 3′ end linkage between two or more of the polynucleotides such that the MNA compound comprises two or more 5′ ends. In some embodiments, one or more of the 5′ ends is modified to facilitate stability of the MNA. In certain embodiments, the two or more polynucleotides linked via the at least one 3′ end linkage each are non-coding nucleotides. In some embodiments, a MNA comprises a synthetic polyribonucleotide or polydeoxyribonucleotide that does not code for a polypeptide, protein, enzyme, antibody, or receptor. In some embodiments, MNA comprising a synthetic polyribonucleotide or polydeoxyribonucleotide inhibits gene expression. In some embodiments, a synthetic polyribonucleotide of the invention that inhibits gene expression is a small interfering ribonucleic acid (siRNA), a microRNA (miRNA), or a short hairpin RNA (shRNA).

While the administration of exogenous polynucleotides (e.g. DNA or RNA) represents a meaningful advancement for the treatment of diseases, the administration of such exogenous polynucleotides is often hampered by the limited stability of such polynucleotides, particularly following their in vivo administration. For example, following their administration to a subject, many polynucleotides may be subject to nuclease (e.g. exonuclease and/or endonuclease) degradation. Nuclease degradation may negatively influence the capability of a polynucleotide to reach a target cell or to be transcribed and/or translated, the result of which is to preclude the exogenous polynucleotide from exerting an intended therapeutic effect.

In some embodiments, the MCNA of the present invention exhibit increased in vivo stability compared to a single polynucleotide not linked to another polynucleotide by its 3′ end (hereinafter “monomeric polynucleotide”). In some embodiments, the MCNA of the present invention, when delivered in vivo, lead to enhanced protein production compared to a monomeric polynucleotide encoding the same protein. In some embodiments, the MCNA of the present invention, when delivered to a subject, are tolerated better by the subject compared to a corresponding monomeric polynucleotide.

Multimeric Coding Nucleic Acids (MCNA)

In some embodiments, the present invention provides compositions comprising multimeric coding nucleic acids (MCNA) and methods for synthesizing the same. In particular, the present invention provides MCNA compounds comprising two or more encoding polynucleotides linked via their 3′ ends such that the MCNA compound comprises two or more 5′ ends and methods of synthesizing the same. In some embodiments, each of the two or more encoding polynucleotides is a synthetic polyribonucleotide. In some embodiments, each of the two or more encoding polynucleotides is a synthetic polydeoxyribonucleotide. In some embodiments, each of the two or more encoding polynucleotides is a synthetic polydeoxyribonucleotide or a polyribonucleotide. In some embodiments, each of the two or more encoding polynucleotides encodes a protein of interest. In some embodiments, each of the two or more encoding polynucleotides encodes a same protein. In some embodiments, each of the two or more encoding polynucleotides encodes a distinct protein. In some embodiments, each of the two or more encoding polynucleotides encoding a distinct protein are present in equal numbers. In some embodiments, each of the two or more encoding polynucleotides encoding a distinct protein are present in unequal numbers (e.g., 2 copies of a polynucleotide encoding protein of interest #1 and 1 copy of a polynucleotide encoding protein of interest #2). In some embodiments, a MCNA compound comprises three or more encoding polynucleotides. In some embodiments, a MCNA compound comprises four or more encoding polynucleotides. In some embodiments, a MCNA compound comprises five or more encoding polynucleotides.

In some embodiments, the present invention provides a multimeric nucleic acid (MNA) comprising two or more polynucleotides linked via at least one 3′ end linkage between two or more of the polynucleotides such that the MNA compound comprises two or more 5′ ends. In some embodiments, one or more of the 5′ ends is modified to facilitate stability of the MNA. In certain embodiments, at least one of the two or more polynucleotides linked via the at least one 3′ end linkage is an encoding polynucleotide and at least one of the two or more polynucleotides linked via the at least one 3′ end linkage is a non-coding polynucleotide, thereby constituting a multimeric coding nucleic acid (MCNA). In certain embodiments, the encoding polynucleotide encodes a protein of interest and the non-coding polynucleotide inhibits gene expression (e.g., small interfering ribonucleic acid (siRNA), a microRNA (miRNA), or a short hairpin RNA (shRNA).

In some embodiments, a MCNA compound comprising two or more encoding polynucleotides encodes one or more chains of an antibody or antibody fragment. In some embodiments, the two or more encoding polynucleotides encode a heavy chain and light chain of an antibody. In some embodiments, the antibody is an intact immunoglobulin, (Fab)2, (Fab′)2, Fab, Fab′ or scFv. In some embodiments, the antibody is an IgG. In some embodiments, the antibody is selected from the group consisting of anti-CCL2, anti-lysyl oxidase-like-2 (LOXL2), anti-Flt-1, anti-TNF-α, anti-Interleukin-2Rα receptor (CD25), anti-TGFβ, anti-B-cell activating factor, anti-alpha-4 integrin, anti-BAGE, anti-β-catenin/m, anti-Bcr-abl, anti-CS, anti-CA125, anti-CAMEL, anti-CAP-1, anti-CASP-8, anti-CD4, anti-CD19, anti-CD20, anti-CD22, anti-CD25, anti-CDC27/m, anti-CD 30, anti-CD33, anti-CD52, anti-CD56, anti-CD80, anti-CDK4/m, anti-CEA, anti-CT, anti-CTL4, anti-Cyp-B, anti-DAM, anti-EGFR, anti-ErbB3, anti-ELF2M, anti-EMMPRIN, anti-EpCam, anti-ETV6-AML1, anti-HER2, anti-G250, anti-GAGE, anti-GnT-V, anti-Gp100, anti-HAGE, anti-HER-2/neu, anti-HLA-A*0201-R1701, anti-IGF-1R, anti-IL-2R, anti-IL-S, anti-MC1R, anti-myosin/m, anti-MUC1, anti-MUM-1, -2, -3, anti-proteinase-3, anti-p190 minor bcr-abl, anti-Pml/RARa, anti-PRAMS, anti-PSA, anti-PSM, anti-PSMA, anti-RAGE, anti-RANKL, anti-RU1 or RU2, anti-SAGE, anti-SART-1 or anti-SART-3, anti-survivin, anti-TEL/AML1, anti-TPI/m, anti-TRP-1, anti-TRP-2, anti-TRP-2/INT2, and anti-VEGF or anti-VEGF receptor.

In some embodiments, a MCNA compound comprising two or more encoding polynucleotides encodes one or more nucleases. In some embodiments, each of the one or more nucleases is selected from the group comprising Cas9, zinc-finger nucleases (ZFN), TALEN, homing endonucleases, homing meganucleases, and combinations thereof. Exemplary nucleases include Afu Uracil-DNA Glycosylase (UDG), Tina Endonuclease III, Tth Endonuclease IV, Antarctic Thermolabile UDG, APE 1, Cas9 Nuclease NLS (S. pyogenes), Cas9 Nuclease (S. pyogenes), DNase I, Endonuclease IV, Endonuclease V, Endonuclease VIII, Exonuclease I, Exonuclease III (E. coli), Exonuclease T, Exonuclease V (RecBCD), Exonuclease VII, Exonuclease VIII (truncated), Fpg, hAAG, hOGG1, hSMUG1, Lambda Exonuclease, Micrococcal Nuclease, Mung Bean Nuclease, Nuclease BAL-31, RecAf, RecJf, T4 PDG (T4 Endonuclease V), T5 Exonuclease, T7 Endonuclease I, T7 Exonuclease, Thermostable FEN1, Uracil Glycosylase Inhibitor (UGI). Exemplary homing nucleases include I-AabMI, I-AniI, I-CeuI, I-CkaMI, I-CpaMI, I-CreI, I-DmoI, I-GpeMI, I-GpiI, I-GzeI, I-GzeII, I-HjeMI, I-LtrI, I-LtrWI, I-MpeMI, I-MsoI, I-OnuI, I-PanMI, I-SceI, I-SmaMI, I-Vdi141I, PI-SceI, I-CreI (m), I-MsoI (m), I-OnuI (E2), I-AniI/I-OnuI, I-DmoI/I-CreI, I-GpiI/I-OnuI, I-GzeI/I-PanMI, I-LtrI/I-PanMI, I-OnuI/I-LtrI, I-AaeMIP, I-ApaMIP, I-GzeMIIIP. I-NcrMIP, I-OsoMIIP, I-PanMIIIP, I-PanMIIP, I-ScuMIIIP, I-ScuMIIP, I-ScuMIP, and I-ScuMIVP.

In some embodiments, a MCNA compound comprises two more polynucleotides that include one, two, or more encoding polynucleotides, wherein each encoding polynucleotide comprises a polynucleotide portion that is an mRNA transcript for a gene and/or for a protein selected from Table 1, Table 2, Table 3, Table 4, Table 5 or Table 6.

TABLE 1 DISEASE/DISORDERS GENE(S) Neoplasia PTEN; ATM; ATR; EGFR; ERBB2; ERBB3; ERBB4; Notch1; Notch2; Notch3; Notch4; AKT; AKT2; AKT3; HIF; H1Fla; HIF3a; Met; HRG; Bcl2; PPARalpha; PPAR gamma; WT1 (Wilms Tumor); FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB (retinoblastoma); MEN!; VHL; BRCA1; BRCA2; AR (Androgen Receptor); TSG101; IGF; IGF Receptor; IgfI (4 variants); Igf2 (3 variants); IgfI Receptor; Igf2 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12); Kras; Apc Age-related Macular Aber; Ccl2; Cc2; cp (ceruloplasmin); Timp3; cathepsinD; Vldlr; Degeneration Ccr2 Schizophrenia Disorders NeuregulinI (NrgI); Erb4 (receptor for Neuregulin); ComplexinI (CplxI); TphI Tryptophan hydroxylase; Tph2 Tryptophan hydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b; 5-HIT (Slc6a4); COMT; DRD (DrdIa); SLC6A3; DAOA; DTNBPI; Dao (Dao1) Trinucleotide Repeat HTT (Huntington's Dx); SBMA/SMAXI/AR (Kennedy's Dx); Disorders FXN/X25 (Friedrich's Ataxia); ATX3 (Machado-Joseph's Dx); ATXNI and ATXN2 (spinocerebellar ataxias); DMPK (myotonic dystrophy); Atrophin-1 and Atn1(DRPLA Dx); CBP (Creb-BP-global instability); VLDLR (Alzheimer's); Atxn7; Atxn10 Fragile X Syndrome FMR2; FXRI; FXR2; mGLUR5 Secretase Related APH-1 (alpha and beta); Presenilin (Psen1); nicastrin Disorders (Ncstn); PEN-2 Others Nos1; Parp1; Nat1; Nat2 Prion-related Disorders Prp ALS SOD1; ALS2; STEX; FUS; TARD BP; VEGF (VEGF-a; VEGF-b; VEGF-c) Drug Addiction Prkce (alcohol); Drd2; Drd4; ABAT (alcohol); GRIA2; Grm5; Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol) Autism Mecp2; BZRAP1; MDGA2; Sema5A; Neurexin 1; Fragile X (FMR2 (AFF2); FXR1; FXR2; Mglur5) Alzheimer's Disease E1; CHIP; UCH; UBB; Tau; LRP; PICALM; Clusterin; PS1; SORL1; CR1; Vld1r; Uba1; Uba3; CHIP28 (Aqp1, Aquaporin 1); Uchl1; Uchl3; APP Inflammation IL-10; IL-1 (IL-Ia; IL-Ib); IL-13; IL-17 (IL-17a (CTLA8); IL-17b; IL- 17c; IL-17d; IL-171); 11-23; Cx3crl; ptpn22; TNFa; NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4; Cx3cll Parkinson's Disease x-Synuclcin; DJ-1; LRRK2; Parkin; PINK1

TABLE 2 CELLULAR FUNCTION GENES Blood and Anemia (CRAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, coagulation diseases PSNI, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, and disorders ABCB7, ABC7, ASAT); Bare lymphocyte syndrome (TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5), Bleeding disorders (TBXA2R, P2RX1, P2X1); Factor Hand factor H-like 1 (HF1, CFH, HUS); Factor V and Factor VIII (MCFD2); Factor VII deficiency (F7); Factor X deficiency (FIO); Factor XI deficiency (F11); Factor XII deficiency (F12, HAF); Factor XIIIA deficiency (F13AI, F13A); Factor XIIIB deficiency (F13B); Fanconi anemia (FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCDI, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BR1PI, BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596); Hemophagocytic lymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3); Hemophilia A (F8, FSC, HEMA); Hemophilia B (F9, HEMB), Hemorrhagic disorders (PI, ATT, F5); Leukocyte deficiencies and disorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia (HBA2, HBB, HBD, LCRB, HBA1). Cell dysregulation B-cell non-Hodgkin lymphoma (BCL7A, BCL7); Leukemia (TALI, and oncology TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1AI, 1KI, LYF1, diseases and disorders HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AFIO, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPMI, NUP214, D9S46E, CAN, CAIN, RUNXI, CBFA2, AML1, WHSC1LI, NSD3, FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF1Q, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPNII, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABLI, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN). Inflammation and AIDS (KIR3DL1, NKAT3, NKB1, AMB11, K1R3DS1, IFNG, CXCL12, immune related SD F1); Autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, diseases and disorders FAS, CD95, ALPS1A); Combined immunodeficiency, (IL2RG, SCIDX1, SCIDX, IMD4); HN-1 (CCL5, SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficienies (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSFS, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI; Inflammation (IL-10, IL-1 (IL-Ia, IL-Ib), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), 11-23, Cx3crI, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cII); Severe combined immunodeficiencies (SCIDs)(JAK3, JAKL, DCLREIC, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDXI, SCIDX, IMD4). Metabolic, liver, Amyloid neuropathy (TTR, PALB); Amyloidosis (APOA1, APP, AAA, kidney and protein CVAP, AD1, GSN, FGA, LYZ, TTR, PALB); Cirrhosis (KRT18, KRT8, diseases and disorders CIRH1A, NAIC, TEX292, KIAA1988); Cystic fibrosis (CFTR, ABCC7, CF, MRP7); Glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM); Hepatic adenoma, 142330 (TCF1, HNF1A, MODY3), Hepatic failure, early onset, and neurologic disorder (SCOD1, SCO1), Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancer and carcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS, AX1NI, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; Medullary cystic kidney disease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2); Phenylketonuria (PAH, PKU1, QDPR, DHPR, PTS); Polycystic kidney and hepatic disease (FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63). Muscular/skeletal Becker muscular dystrophy (DMD, BMD, MYF6), Duchenne Muscular diseases and disorders Dystrophy (DMD, BMD); Emery-Dreifuss muscular dystrophy (LMNA, LMN1, EMD2, FPLD, CMDIA, HGPS, LGMDIB, LMNA, LMNI, EMD2, FPLD, CMD1A); Facioscapulohumeral muscular dystrophy (FSHMD1A, FSHD1A); Muscular dystrophy (FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDCIC, LGMD21, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1); Osteopetrosis (LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTMI, GL, TCIRG1, TIRC7, OC116, OPTB1); Muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1). Neurological and ALS (SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, neuronal diseases VEGF-c); Alzheimer disease (APP, AAA, CVAP, AD1, APOE, AD2, and disorders PSEN2, AD4, STM2, APBB2, FE65LI, NOS3, PLAU, URK, ACE, DCPI, ACEI, MPO, PAC1PI, PAXIPIL, PTIP, A2M, BLMH, BMH, PSEN1, AD3); Autism (Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2, mGLUR5); Huntington's disease and disease like disorders (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17); Parkinson disease (NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN, PARK2, PDJ, DBH, NDUFV2); Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x-Synuclein, DJ-1); Schizophrenia (Neuregulin1 (Nrg1), Erb4 (receptor for Neuregulin), Complexin1 (Cplx1), Tph1 Tryptophan hydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), CONT, DRD (Drd1a), SLC6Aβ, DAOA, DTNBP1, Dao (Dao1)); Secretase Related Disorders (APH-1 (alpha and beta), Presenilin (Psen1), nicastrin, (Ncstn), PEN-2, Nos1, Parp1, Nat1, Nat2); Trinucleotide Repeat Disorders (HTT (Huntington's Dx), SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich's Ataxia), ATX3 (Machado-Joseph's Dx), ATXN1 and ATXN2 (spinocerebellar ataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP (Creb-BP-global instability), VLDLR (Alzheimer's), Atxn7, Atxn10). Occular diseases Age-related macular degeneration (Aber, Ccl2, Cc2, cp (ceruloplasmin), and disorders Timp3, cathepsinD, Vldlr, Ccr2); Cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYAI, PAX6, AN2 MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQPO, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1); Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1SI, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma (MYOC, TIGR, GLCIA, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1BI, GLC3A, OPA1, NTG, NPG, CYP1BI, GLC3A); Leber congenital amaurosis (CRB1, RP12, CRX, CORD2, CRD, RPGRIPI, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy (ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2). Epilepsy NHLRC1, EPM2A, EPM2B Duchenne muscular DMD, BMD dystrophy AIDS KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CDDCL12, SDF1 Alpha 1-Antitrypsin SERPINA1 [serpin peptidase inhibitor, cladeA (alpha-1 Deficiency antiproteinase, antitrypsin), member 1]; SERPINA2 [serpin peptidase inhibitor, cladeA (alpha-1 antiproteinase, antitrypsin), member 2]; SERPINA3 [serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3]; SERPINA5 [serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 5]; SERPINA6 [serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 6]; SERPINA7 [serpin peptidase inhibitor, Glade A (alpha-1 antiproteinase, antitrypsin), member 7]; SERPINA6 (serpin peptidase inhibitor, cladeA (alpha-1 antiproteinase, antitrypsin), member 6)

TABLE 3 CELLULAR FUNCTION GENES PI3K/AKT Signaling PRKCE; ITGAM; ITGA5; IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E; PRKCZ; GRK6: MAPK1; TSC1; PLK1; AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; PIK3CB; PPP2R1A; MAPK8; BCL2LI; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1; MAPK9; CDK2; PPP2CA; PIM!; ITGB7; YWHAZ; ILK; TP53; RAF!; IKBKG; RELB; DYRK1A; CDKNIA; ITGB1; MAP2K2; JAK1; AKT1; JAK2; PIK3RI; CHUK; PDPK1; PPP2R5C; CTNNB1; MAP2K1; NFKB1; PAK3; ITGB3; CCND1; GSK3A; FRAP!; SFN; ITGA2; TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP90AA1; RPS6KB1 ERK/MAPK Signaling PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2; RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; CREB1; PRKC1; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3; MAPK8; MAPK3; ITGA1; ETSI; KRAS; MYCN; EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9; SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSR1; PXN; RAF!; FYN; DYRK1A; ITGB1; MAP2K2; PAK4; PIK3RI; STAT3; PPP2R5C; MAP2KI; PAK3; ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF; STAT1; SGK Glucocorticoid Receptor RAC1; TAF4B; EP300; SMAD2; TRAF6; PCAF; ELK1; Signaling MAPKI; SMAD3; AKT2; IKBKB; NCOR2; UBE21; PIK3CA; CREBI; FOS; HSPA5; NFKB2; BCL2; MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8; BCL2L1; MAPK3; TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; RELA; STAT5A; MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3; MAPK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; IL8; NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; ESR1; SMAD4; CEBPB; WN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1 Axonal Guidance PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12; Signaling IGF1; RAC1; RAP1A; EIF4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1; GNAQ; PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; ADAM17; AKT1; PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA Ephrin Receptor PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1; AKT2; DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14; CXCL12; MAPK8; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2; PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4, AKT1; JAK2; STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK; CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK Actin Cytoskeleton ACTN4; PRKCE; ITGAM; ROCK1; ITGA5; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1; INS; ARHGEF7; GRK6; ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CPL1; PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN; VIL2; RAF1; GSN; DYRK1A; ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1; PAK3; ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGK Huntington's Disease PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2; Signaling MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2; PIK3CA; HDAC5; CREB1; PRKCI; HSPA5; REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1; GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A; PRKCD; HDAC11; MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; PDPK1; CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4; AKT3; PRKCA; CLTC; SGK; HDAC6; CASP3 Apoptosis Signaling PRKCE; ROCK1; BID; IRAK1; PRKAA2; EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2L1; CAPN1; MAPK3; CASP8; KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG; RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA; CASP2; BIRC2; TTK; CSNKIA1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1 B Cell Receptor RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; Signaling AKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3; MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9; EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN; GSK3B; ATF4; AKT3; VAV3; RPS6KB1 Leukocyte Extravasation ACTN4; CD44; PRKCE; ITGAM; ROCK1; CXCR4; CYBA; Signaling RAC1; RAP1A; PRKCZ; ROCK2; RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8; PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A; BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1; PIK3R1; CTNNB1; CLDN1; CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9 Integrin Signaling ACTN4; ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1; ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3; MAPK8; CAV1; CAPN1; ABL1; MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7; PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1; TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3 Acute Phase Response IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11; Signaling AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8; RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1; TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3; IL1R1; IL6 PTEN Signaling ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11; MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2; PIK3CB; BCL2L1; MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1; IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1; MAP2K1; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1; CASP3; RPS6KB1 p53 Signaling PTEN; EP300; BBC3; PCAF; FASN; BRCA1; GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3; MAPK8; THBS1; ATR; BCL2L1; E2F1; PMAIP1; CHEK2; TNFRSF10B; TP73; RB1; HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1; RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2; GSK3B; BAX; AKT3 Aryl Hydrocarbon HSPB1; EP300; FASN; TGM2; RXRA; MAPK1; NQO1; Receptor Signaling NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1; SMARCA4; NEKB2; MAPK8; ALDH1A1; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73; GSTP1; RB1; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2; APAF1; NFKB1; CCND1; ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX; IL6; CYP1B1; HSP90AA1 Xenobiotic Metabolism PRKCE; EP300; PRKCZ; RXRA; MAPK1; NQO1; Signaling NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB; PPP2R1A; PIK3C3; MAPK8; PRKD1; ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13; PRKCD; GSTP1; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A; PPARGC1A; MAPK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1; NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AA1 SAPK/JNK Signaling PRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2; PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1; IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3; CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK PPAr/RXR Signaling PRKAA2; EP300; INS; SMAD2; TRAF6; PPARA; FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB; NCOR2; ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8; IRS1; MAPK3; KRAS; RELA; PRKAA1; PPARGC1A; NCOA3; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1; ADIPOQ NF-KB Signaling IRAK1; EIF2AK2; EP300; INS; MYD88; PRKCZ; TRAF6; TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2; MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A; TRAF2; TLR4: PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1; PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1 Neuregulin Signaling ERBB4; PRKCE; ITGAM; ITGA5; PTEN; PRKCZ; ELK1; MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3; ITGA1; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM! 7; AKT1; PIK3RI; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC; NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Wnt & Beta catenin CD44; EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; Signaling AKT2; PIN1; CDH1; BTRC; GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2: ILK; LEF1; SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1; TGFBR1; CCND1; GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2 Insulin Receptor PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; Signaling PTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPKS; IRS1; MAPK3; TSC2; KRAS; EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2; JAK1; AKT1; JAK2; PIK3RI; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B; AKT3; FOXO1; SGK; RPS6KB1 IL-6 Signaling HSPB1; TRAF6; MAPKAPK2; ELK1; MAPK1; PTPN11; IKBKB; FOS; NFKB2: MAP3K14; MAPKS; MAPK3; MAPK10; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2KI; NFKB1; CEBPB; JUN; IL1R1; SRF; IL6 Hepatic Cholestasis PRKCE; IRAK1; INS; MYDSS; PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPKS; PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG; RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKB1; ESR1; REBF1; FGFR4; JUN; IL1R1; PRKCA; IL6 IGF-1 Signaling IGF1; PRKCZ; ELK1; MAPK1; PTPN11; NEDD4; AKT2; PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPKS; IGF1R; IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXO1; SRF; CTGF; RPS6KB1 NRF2-mediated PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1; Oxidative NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MAPK8; Stress Response PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A; MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN; KEAP1; GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 Hepatic Fibrosis/Hepatic EDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF; Stellate Cell Activation SMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9; IGF1R; IL6R; RELA; TLR4; PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1; CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 PPAR Signaling EP300; INS; TRAF6; PPARA; RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B; MAPK3; NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF; INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2KI; NFKB1; JUN; IL1R1; HSP90AA1 Fc Epsilon R1 Signaling PRKCE; RAC1; PRKCZ; LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK; MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1; PIK3RI; PDPK1; MAP2K1; AKT3; VAV3; PRKCA G-Protein Coupled PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB; Receptor Signaling PIK3CA; CREB1; GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3; MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1; PIK3R1; CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCA Inositol Phosphate PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; Metabolism MAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1; MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK PDGF Signaling EIF2AK2; ELK1; ABL2; MAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3; KRAS; SRC; PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA; STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGF Signaling ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA; ARNT; PTK2; BCL2; PIK3CB; PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3; PIK3C2A; PXN; RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA; AKT3; FOXO1; PRKCA Natural Killer Cell PRKCE; RAC1; PRKCZ; MAPK1; RAC2; PTPN11; Signaling KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; PRKD1; MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4; AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: G1/S HDAC4; SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; Checkpoint Regulation ATR; ABL1; E2F1; HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53; CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1; HDAC6 T Cell Receptor RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS; Signaling NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA; PIK3C2A; BTK; LCK; RAF1; IKBKG; RELB; FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK; BCL10; JUN; VAV3 Death Receptor Signaling CRADD; HSPB1; BID; BIRC4; TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX; TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1; CASP2; BIRC2; CASP3; BIRC3 FGF Signaling RAC1; FGFR1; MET; MAPKAPK2; MAPK1; PTPN11; AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3; MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1; FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF GN-CSF Signaling LYN; ELK1; MAPK1; PTPN11; AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1; MAPK3; ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2; PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1 Amyotrophic Lateral BID; IGF1; RAC1; BIRC4; PGF; CAPNS1; CAPN2; Sclerosis Signaling PIK3CA; BCL2; PIK3CB; PIK3C3; BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A; CASP1; APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/Stat Signaling PTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS; SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STAT1 Nicotinate and PRKCE; IRAK1; PRKAA2; EIF2AK2; GRK6; MAPK1; Nicotinamide LK1; AKT2; T2; CDK8; MAPK8; MAPK3; PRKCD; PRKAA1; Metabolism PBEF1; MAPK9; CDK2; PIMI; DYRK1A; MAP2K2; MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine Signaling CXCR4; ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8; MAPK3; KRAS; MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1; MAP2K2; MAP2K1; JUN; CCL2; PRKCA IL-2 Signaling ELK1; MAPK1; PTPN11; AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1; JUN; AKT3 Synaptic Long Term PRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1; GNAS; Depression PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCA Estrogen Receptor TAF4B; EP300; CARM1; PCAF; MAPK1; NCOR2; Signaling SMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1; HDAC3; PPARGG1A; RBM9; NCOA3; RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 Protein Ubiquitination TRAF6; SMURF1; BIRC4; BRCA1; UCHL1; NEDD4; Pathway CBL; UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; USP1; VHL; HSP90AA1; BIRC3 IL-10 Signaling TRAF6; CCR1; ELK1; IKBKB; SP1; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF; IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6 VDR/RXR Activation PRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1; PRKCI; CDKN1B; PRKD1; PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1; LRP5; CEBPB; FOXO1; PRKCA TGF-beta Signaling EP300; SMAD2; SMURF1; MAPK1; SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS; MAPK9; RUNX2; SERPINE1; RAF1; MAP3K7; CREBBP; MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5 Toll-like Receptor IRAK1; EIF2AK2; MYD88; TRAF6; PPARA; ELK1; Signaling IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN P38 MAPK Signaling HSPB1; IRAK1; TRAF6; MAPKAPK2; ELK1; FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2; MAPK14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF; STAT1 Neurotrophin/TRK NTRK2; MAPK1; PTPN11; PIK3CA; CREB1; FOS; Signaling PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; CDC42; JUN; ATF4 FXR/RXR Activation INS; PPARA; FASN; RXRA; AKT2; SDC1; MAPK8; APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A; TNF; CREBBP; AKT1; SREBF1; FGFR4; AKT3; FOXO1 Synaptic Long Term PRKCE; RAP1A; EP300; PRKCZ; MAPK1; CREB1; Potentiation PRKCI; GNAQ; CAMK2A; PRKD1; MAPK3; KRAS; PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4; PRKCA Calcium Signaling RAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1; CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4; HDAC6 EGF Signaling ELK1; MAPK1; EGFR; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3; PIK3C2A; RAF1; JAK1; PIK3R1; STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1 Hypoxia Signaling in the EDN1; PTEN; EP300; NQO1; UBE2I; CREB1; ARNT; Cardiovascular System HIF1A; SLC2A4; NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN; ATF4; VHL; HSP90AA1 LPS/IL-1 Mediated IRAK1; MYD88; TRAF6; PPARA; RXRA; ABCA1; Inhibition MAPK8; ALDH1A1; GSTP1; MAPK9; ABCB1; TRAF2; of RXR Function TLR4; TNF; MAP3K7; NR1H2; SREBF1; JUN; IL1R1 LXR/RXR Activation FASN; RXRA; NCOR2; ABCA1; NFKB2; IRF3; RELA; NOS2A; TLR4; TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1; IL1R1; CCL2; IL6; MMP9 Amyloid Processing PRKCE; CSNK1E; MAPK1; CAPNS1; AKT2; CAPN2; CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1; GSK3B; AKT3; APP IL-4 Signaling AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1; KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1; AKT3; RPS6KB1 Cell Cycle: G2/M DNA EP300; PCAF; BRCA1; GADD45A; PLK1; BTRC; Damage Checkpoint CHEK1; ATR; CHEK2; YWHAZ; TP53; CDKN1A; Regulation PRKDC; ATM; SFN; CDKN2A Nitric Oxide Signaling in KDR; FLT1; PGF; AKT2; PIK3CA; PIK3CB; PIK3C3; the Cardiovascular System CAV1; PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1; VEGFA; AKT3; HSP90AA1 Purine Metabolism NME2; SMARCA4; MYH9; RRM2; ADAR; EIF2AK4; PKM2; ENTPD1; RAD51; RRM2B; TJP2; RAD51C; NT5E; POLD1; NME1 cAMP-mediated RAP1A; MAPK1; GNAS; CREB1; CAMK2A; MAPK3; Signaling SRC; RAF1; MAP2K2; STAT3; MAP2K1; BRAF; ATF4 Mitochondrial SOD2; MAPK8; CASP8; MAPK10; MAPK9; CASP9; Dysfunction PARK7; PSEN1; PARK2; APP; CASP3 Notch Signaling HES1; JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3; NOTCH1; DLL4 Endoplasmic Reticulum HSPA5; MAPK8; XBP1; TRAF2; ATF6; CASP9; ATF4; Stress Pathway EIF2AK3; CASP3 Pyrimidine Metabolism NME2; AICDA; RRM2; EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Parkinson's Signaling UCHL1; MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2; CASP3 Cardiac & Beta GNAS; GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; Adrenergic Signaling PPP2R5C Glycolysis/ HK2; GCK; GPI; ALDH1A1; PKM2; LDHA; HK1 Gluconeogenesis Interferon Signaling IRF1; SOCS1; JAK1; JAK2; IFITM1; STAT1; IFIT3 Sonic Hedgehog ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK39; DYRK1B Signaling Glycerophospholipid PLD1; GRN; GPAM; YWHAZ; SPHK1; SPHK2 Metabolism Phospholipid PRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2 Degradation Tryptophan Metabolism SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1; SIAH1 Lysine Degradation SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C Nucleotide Excision ERCC5; ERCC4; XPA; XPC; ERCC1 Repair Pathway Starch and Sucrose UCHL1; HK2; GCK; GPI; HK1 Metabolism Aminosugars Metabolism NQO1; HK2; GCK; HK1 Arachidonic Acid PRDX6; GRN; YWHAZ; CYP1B1 Metabolism Circadian Rhythm CSNK1E; CREB1; ATF4; NR1D1 Signaling Coagulation System BDKRB1; F2R; SERPINE1; F3 Dopamine Receptor PPP2R1A; PPP2CA; PPP1CC; PPP2R5C Signaling Glutathione Metabolism IDH2; GSTP1; ANPEP; IDH1 Glycerolipid Metabolism ALDH1A1; GPAM; SPHK1; SPHK2 Linoleic Acid Metabolism PRDX6; GRN; YWHAZ; CYP1B1 Methionine Metabolism DNMT1; DNMT3B; AHCY; DNMT3A Pyruvate Metabolism GLO1; ALDH1A1; PKM2; LDHA Arginine and Proline ALDH1A1; NOS3; NOS2A Metabolism Eicosanoid Signaling PRDX6; GRN; YWHAZ Fructose and Mannose HK2; GCK; HK1 Metabolism Galactose Metabolism HK2; GCK; HK1 Stilbene, Coumarine and PRDX6; PRDX1; TYR Lignin Biosynthesis Antigen Presentation CALR; B2M Pathway Biosynthesis of Steroids NQO1; DHCR7 Butanoate Metabolism ALDH1A1; NLGN1 Citrate Cycle IDH2; IDH1 Fatty Acid Metabolism ALDH1A1; CYP1B1 Glycerophospholipid PRDX6; CHKA Metabolism Histidine Metabolism PRMT5; ALDH1A1 Inositol Metabolism ERO1L; APEX1 Metabolism of GSTP1; CYP1B1 Xenobiotics by Cytochrome p450 Methane Metabolism PRDX6; PRDX1 Phenylalanine PRDX6; PRDX1 Metabolism Propanoate Metabolism ALDH1A1; LDHA Selenoamino Acid PRMT5; AHCY Metabolism Sphingolipid Metabolism SPHK1; SPHK2 Aminophosphonate PRMT5 Metabolism Androgen and Estrogen PRMT5 Metabolism Ascorbate and Aldarate ALDH1A1 Metabolism Bile Acid Biosynthesis ALDH1A1 Cysteine Metabolism LDHA Fatty Acid Biosynthesis FASN Glutamate Receptor GNB2L1 Signaling NRF2-mediated PRDX1 Oxidative Stress Response Pentose Phosphate GPI Pathway Pentose and Glucuronate UCHL1 Interconversions Retinol Metabolism ALDH1A1 Riboflavin Metabolism TYR Tyrosine Metabolism PRMT5, TYR Ubiquinone Biosynthesis PRMT5 Valine, Leucine and ALDH1A1 Isoleucine Degradation Glycine, Serine and CHKA Threonine Metabolism Lysine Degradation ALDH1A1 Pain/Taste TRPM5; TRPA1 Pain TRPM7; TRPC5; TRPC6; TRPC1; Cnr1; cnr2; Grk2; Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5; Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial Function AIF; CytC; SMAC (Diablo); Aifm-1; Aifm-2 Developmental BMP-4; Chordin (Chrd); Noggin (Nog); WNT (Wnt2; Neurology Wnt2b, Wnt3a, Wnt4; Wnt5a; Wnt6; Wnt7b; Wnt8b; Wnt9a; Wnt9b; Wnt10a; Wnt10b; Wnt16); beta-catenin; Dkk-1; Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1; unc-86 (Pou4f1 or Brn3a); Numb; Reln

TABLE 4 INDICATION(S) THERAPEUTIC PROTEIN Maple syrup urine disease 3-methyl-2-oxobutanoate dehydrogenase Medium-chain acyl-CoA Acyl-CoA dehydrogenase dehydrogenase deficiency Alpha 1-antitrypsin deficiency Alpha 1 protease inhibitor Pompe disease Alpha glucosidase Paroxysmal nocturnal Anti-complement factor C5 Mab hemoglobinuria Familial dysbetalipoproteinemia Apolipoprotein E Argininemia Arginase Argininosuccinic acidemia Argininosuccinate lyase Citrullinemia, type I Argininosuccinate synthase Short-chain acyl-CoA Butyryl-CoA dehydrogenase dehydrogenase deficiency Hereditary angioedema C1 esterase inhibitor Carbamylphosphate synthetase Carbamylphosphate synthetase deficiency Cystic fibrosis CFTR Hemophilia B Factor IX Hemophilia A, Hemophilia B Factor VII Hemophilia A Factor VIII Classical galactosemia Galactose-1-phosphate uridylyltransferase von Gierke's disease Glucose-6-phosphatase Glutaric acidemia, type I Glutaryl-CoA dehydrogenase Isovaleric aciduria Isovaleric acid CoA dehydrogenase deficiency Homozygous familial LDL receptor hypercholesterolemia Long-chain 3-OH acyl-CoA Long-chain-3-hydroxyacyl-CoA dehydrogenase dehydrogenase deficiency Very long-chain acyl-CoA Long-chain-acyl-CoA dehydrogenase dehydrogenase deficiency Methylmalonyl-CoA mutase Methylmalonyl-CoA mutase deficiency Ornithine transcarbamylase Ornithine transcarbamylase deficiency Phenylketonuria Phenylalanine hydroxylase Acute intermittent porphyria Porphobilinogen deaminase Propionic acidemia Propionyl-CoA carboxylase Hyperoxaluria, type I Serine-pyruvate aminotransferase Crigler-Najjar syndrome UDP-glucuronosyltransferase Non-Hodgkin lymphoma Anti-CD20 mAb Allergic asthma Anti-IgE mAb Psoriasis Anti-IL-12 & IL-23 mAb Rheumatoid arthritis Anti-interleukin-6 (IL-6) mAb Anemia Erythropoietin Rheumatoid arthritis T-cell costimulation blocker Rheumatoid arthritis TNF-alpha inhibitors (including anti-TNF-alpha mAb) Gout Urate oxidase Familial chylomicronemia Lipoprotein lipase Melanoma Anti-CTLA4 mAb Head and neck cancer, Metastatic Anti-EGFr mAb colorectal cancer HER2+ breast cancer, gastric Anti-HER2 mAb cancer Metastatic colorectal cancer, Anti-VEGF mAb NSCLC, others Blepharospasm, Cervical Botulinum toxin dystonia, Chronic migraine, more Female infertility Follicle stimulating hormone Type 2 diabetes mellitus Glucagon-like peptide 1 (GLP-1) agonist Growth hormone deficiency Growth hormone 1/Growth hormone 2 Type 2 diabetes mellitus Insulin Hypoparathyroidism Parathyroid hormone Asthma SERCA2 Asthma FoxP3 Surfactant Deficiency Pulmonary surfactants (SFTPA1, SFTPB, SFTPC, SFTPD) Pulmonary Alveolar proteinosis GM-CSF Receptor (CSF2RA, CSF2RB) alport syndrome Col4A5 Stargardt's Disease ABCA4 Retinitis pigmentosa Rhodopsins Adrenoleukodystrophy ABCD1 Adenosine deaminase deficiency Adenosine deaminase Familial adenomatous polyposis APC Autosomal recessive polycystic ARPKD kidney disease Metachromatic leukodystrophy Arylsulfatase A Batten disease Battenin + others Beta-thalassemia Beta globin X-linked agammaglobulinemia Bruton's tyrosine kinase Becker muscular dystrophy Dystrophin Duchenne muscular dystrophy Dystrophin Marfan syndrome FBN1 Fragile X syndrome FMRP Krabbe disease Galactocerebrosidase Sickle cell disease Hemoglobin Sanfilippo syndrome, type A (MPS Heparan N-sulfatase IIIA) GM2 gangliosidosis HEXA, HEXB Hemachromatosis HFE protein Huntington disease Huntingtin Lesch-Nyhan syndrome Hypoxanthine phosphoribosyltransferase 1 McArdle disease Muscle glycogen phosphorylase Sanfilippo syndrome, type B (MPS N-acetyl-alpha-D-glucosaminidase IIIB) Leber's hereditary optic NADH dehydrogenase neuropathy Neurofibromatosis, type 1 NF-1 Niemann Pick disease, type C NPC1 Alpers' disease POLG Von Hippel-Lindau disease pVHL Paget disease of bone Sequestosome 1 Carnitine uptake defect SLC22A5 Cystinuria SLC7A9 Niemann Pick disease, type A/B SMPD1 Spinal muscular atrophy Survival motor neuron protein Li-Fraumeni syndrome TP53 Fabry disease Alpha galactosidase Alpha-mannosidosis Alpha-D-mannosidase Hurler syndrome (MPS I) Alpha-L iduronidase Hemolytic uremic syndrome Anti-complement factor C5 mAb Morquio syndrome, type B (MPS Beta-galactosidase IVB) Multiple carboxylase deficiency Biotin-methylcrotonoyl-CoA-carboxylase ligase Homocystinuria Cystathionine beta-synthase Cystinosis Cystinosin Cystic fibrosis Deoxyribonuclease I Erythropoietic protoporphyria Ferrochelatase Tyrosinemia, type I Fumarylacetoacetase GALK deficiency Galactokinase Morquio syndrome, type A (MPS Galactose 6-sulfate sulfatase IVA) GALE deficiency Galactose epimerase Gaucher disease Glucocerebrosidase Alkaptonuria Homogentisate 1,2-dioxygenase Hunter syndrome (MPS II) Iduronate-2-sulfatase Lysosomal acid lipase deficiency Lysosomal acid lipase Hypermethioninemia Methionine adenosyltransferase 3-Methylcrotonyl-CoA Methylcrotonoyl-CoA carboxylase carboxylase deficiency 3-Methylglutaconic aciduria Methylglutaconyl-CoA hydratase Maroteaux-Lamy syndrome (MPS N-acetylgalactosamine 4-sulfatase VI) Familial mediterranean fever Pyrin (MEFV) Tetrahydrobiopterin-deficient Tetrahydrobiopterin hyperphenylalaninemia Juvenile rheumatoid arthritis TNF-alpha inhibitors Psoriatic arthritis TNF-alpha inhibitors Hypophosphatasia TNSALP Gilbert syndrome UDP-glucuronosyltransferase Porphyria cutanea tarda Uroporphyrinogen decarboxylase Wilson disease Wilson disease protein Systemic lupus erythematosus Anti-BAFF Osteoporosis Anti-RANKL mAb Multiple sclerosis Anti-VLA-4 mAb Neutropenia G-CSF Immunoglobulin deficiency Immunoglobulin Primary humoral immune Immunoglobulin deficiencies (e.g., CVID) Infectious diseases vaccines Infectious antigen Hepatitis B, Hepatitis C Interferon alpha Multiple sclerosis Interferon beta Chronic immune Thrombopoietin thrombocytopenia Ehlers-Danlos syndrome, type 1 Proteins encoded by ADAMTS2, B3GALT6, B4GALT7, CHST14, COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, DSE, FKBP14, PLOD1, PRDM5, SLC39A13, TNXB, and ZNF469 Stickler syndrome Proteins encoded by COL11A1, COL11A2, COL2A1, COL9A1, COL9A2, and COL9A3 Hereditary hemorrhagic Proteins encoded by ACVRL1, ENG, and SMAD4 telangiectasia Hereditary spherocytosis Proteins encoded by ANK1, EPB42, SLC4A1, SPTA1 and SPTB Brugada syndrome Proteins encoded by CACNA1C, CACNA2D1, CACNB2, GPD1L, HCN4, KCND3, KCNE3, KCNE5, KCNJ8, RANGRF, SCN1B, SCN2B, SCN3B, SCN5A, SLMAP, and TRPM4 Osteopetrosis Proteins encoded by CA2, CLCN7, IKBKG, ITGB3, OSTM1, PLEKHM1, TCIRG1, TNFRSF11A, and TNFSF11 Mitochondrial oxidative Proteins encoded by FBXL4, and NDUFB9 phosphorylation disorders

TABLE 5 INDICATION(S) THERAPEUTIC PROTEIN GENE Achromatopsia type 2 Cyclic nucleotide-gated channel, CNGA3 α3 subunit Achromatopsia type 3 Cyclic nucleotide-gated channel, CNGB3 β3 subunit Aland Island eye disease Cav1.4: calcium channel, voltage- CACNA1F gated, L type, α1F subunit Andersen-Tawil syndrome Kir2.1: potassium channel, KCNJ2 inwardly-rectifying, subfamily J, member 2 Benign familial infantile epilepsy Nav2.1: sodium channel, voltage- SCN2A gated, type II, α subunit Kv7.2: potassium channel, KCNQ2 voltage-gated, KQT-like subfamily, member 2 Kv7.3: potassium channel, KCNQ3 voltage-gated, KQT-like subfamily, member 3 Bestrophinopathy, autosomal- Bestrophin 1 BEST1 recessive Central core disease RyR1: ryanodine receptor 1 RYR1 Charcot-Marie-Tooth disease type Transient receptor potential TRPV4 2C cation channel, subfamily V, member 4 Childhood absence epilepsy γ-aminobutyric acid A receptor, GABRA1 α1 subunit γ-aminobutyric acid A receptor, GABRA6 α6 subunit γ-aminobutyric acid A receptor, GABRB3 β3 subunit γ-aminobutync acid A receptor, γ2 GABRG2 subunit Cav3.2: calcium channel, voltage-gated, T type, α1H subunit CACNA1H Cognitive impairment with or Nav1.6: sodium channel, voltage- SCN8A without cerebellar ataxia gated, type VIII, α subunit Cone-rod dystropy, X-linked, type Cav1.4: calcium channel, voltage- CACNA1F 3 gated, L type, α1F subunit Congenital distal spinal muscular Transient receptor potential TRPV4 atrophy cation channel, subfamily V, member 4 Congenital indifference to pain, Nav1.7: Sodium channel, voltage- SCN9A autosomal-recessive gated, type IX, α subunit Congenital myasthenic syndrome Cholinergic receptor, muscle CHRNA1 nicotinic, α1 subunit Cholinergic receptor, muscle CHRNB1 nicotinic, β1 subunit Cholinergic receptor, muscle CHRND nicotinic, δ subunit Cholinergic receptor, muscle CHRNE nicotinic, ε subunit Nav1.4: sodium channel, voltage- SCN4A gated, type IV, α subunit Congenital stationary night Transient receptor potential TRPM1 blindness type 1C cation channel, subfamily M, member 1 Congenital stationary night Cav1.4: calcium channel, voltage- CACNA1F blindness type 2A gated, L type, α1F subunit Deafness, autosomal-dominant, Kv7.4: potassium channel, KCNQ4 type 2A voltage-gated, KQT-like subfamily, member 4 Deafness, autosomal-recessive, Kir4.1: potassium channel, KCNJ10 type 4, with enlarged inwardly-rectifying, subfamily J, vestibular aqueduct member 10 Dravet syndrome Nav1.1: sodium channel, voltage- SCN1A gated, type I, α subunit γ-aminobutyric acid A receptor, γ2 GABRG2 subunit Early infantile epileptic Kv7.2: potassium channel, KCNQ2 encephalopathy type 7 voltage-gated, KQT-like subfamily, member 2 Early infantile epileptic Nav2.1: sodium channel, voltage- SCN2A encephalopathy type 11 gated, type II, α subunit Early infantile epileptic Nav1.6: sodium channel, voltage- SCN8A encephalopathy type 13 gated, type VIII, α subunit Early infantile epileptic KCa4.1: potassium channel, KCNT1 encephalopathy type 14 subfamily T, member 1 EAST/SeSAME syndrome Kir4.1: potassium channel, KCNJ10 inwardly-rectifying, subfamily J, member 10 Episodic ataxia type 1 Kv1.1: potassium channel, KCNA1 voltage-gated, shaker-related subfamily, member 1 Episodic ataxia type 2 Cav2.1: calcium channel, voltage- CACNA1A gated, P/Q type, α1A subunit Episodic ataxia type 5 Cavβ4: calcium channel, voltage- CACNB4 gated, β4 subunit Familial episodic pain syndrome Transient receptor potential TRPA1 cation channel, subfamily A, member 1 Familial hemiplegic migraine type Cav2.1: calcium channel, voltage- CACNA1A 1 gated, P/Q type, α1A subunit Familial hemiplegic migraine type Nav1.1: sodium channel, voltage- SCN1A 3 gated, type I, α subunit Generalized epilepsy with febrile Navβ1: sodium channel, voltage- SCN1B seizures plus (GEFS+) gated, type I, β subunit Nav1.1: sodium channel, voltage- SCN1A gated, type I, α subunit γ-aminobutyric acid A receptor, γ2 GABRG2 subunit Generalized epilepsy with KCa1.1: potassium channel, KCNMA1 paroxysmal dyskinesia calcium-activated, large conductance, subfamily M, α1 subunit Hereditary hyperekplexia Glycine receptor, α1 subunit GLRA1 Glycine receptor, β subunit GLRB Hyperkalemic periodic paralysis Nav1.4: sodium channel, voltage- SCN4A gated, type IV, α subunit Hypokalemic periodic paralysis Cav1.1: calcium channel, voltage- CACNA1S type 1 gated, L type, α1S subunit Hypokalemic periodic paralysis Nav1.4: sodium channel, voltage- SCN4A type 2 gated, type IV, α subunit Juvenile macular degeneration Cyclic nucleotide-gated channel, CNGB3 β3 subunit Juvenile myoclonic epilepsy γ -aminobutyric acid A receptor, GABRA1 α1 subunit Cavβ4: calcium channel, voltage- CACNB4 gated, β4 subunit Malignant hyperthermia RyR1: ryanodine receptor 1 RYR1 susceptibility Cav1.1: calcium channel, voltage- CACNA1S gated, L type, α1S subunit Mucolipidosis type IV TRPML1/mucolipin 1 MCOLN1 Multiple pterygium syndrome, Cholinergic receptor, muscle CHRNA1 lethal type nicotinic, α1 subunit Multiple pterygium syndrome, Cholinergic receptor, muscle CHRND nonlethal type (Escobar variant) nicotinic, δ subunit Cholinergic receptor, muscle CHRNG nicotinic, γ subunit Myotonia congenita, autosomal- CIC-1: chloride channel 1, voltage- CLCN1 dominant (Thomsen disease) gated Myotonia congenita, autosomal- CIC-1: chloride channel 1, voltage- CLCN1 recessive (Becker disease) gated Nocturnal frontal lobe epilepsy Cholinergic receptor, neuronal CHRNA4 type 1 nicotinic, α4 subunit Nocturnal frontal lobe epilepsy Cholinergic receptor, neuronal CHRNB2 type 3 nicotinic, β2 subunit Nocturnal frontal lobe epilepsy Cholinergic receptor, neuronal CHRNA2 type 4 nicotinic, α2 subunit Nocturnal frontal lobe epilepsy KCa4.1: potassium channel, KCNT1 type 5 subfamily T, member 1 Paramyotonia congenita Nav1.4: sodium channel, voltage- SCN4A gated, type IV, α subunit Paroxysmal extreme pain disorder Nav1.7: Sodium channel, voltage- SCN9A gated, type IX, α subunit Potassium-aggravated myotonia Nav1.4: sodium channel, voltage- SCN4A gated, type IV, α subunit Primary erythermalgia Nav1.7: sodium channel, voltage- SCN9A gated, type IX, α subunit Retinitis pigmentosa type 45, Cyclic nucleotide-gated channel, CNGB1 autosomal-recessive β1 subunit Retinitis pigmentosa type 49, Cyclic nucleotide-gated channel, CNGA1 autosomal-recessive α1 subunit Retinitis pigmentosa type 50, Bestrophin 1 BEST1 autosomal-dominant Scapuloperoneal spinal muscular Transient receptor potential TRPV4 atrophy cation channel, subfamily V, member 4 Small fiber neuropathy Nav1.7: sodium channel, voltage- SCN9A gated, type IX, α subunit Spinocerebellar ataxia type 6 Cav2.1: calcium channel, voltage- CACNA1A gated, P/Q type, α1A subunit Spinocerebellar ataxia type 13 Kv3.3: potassium channel, KCNC3 voltage-gated, Shaw-related subfamily, member 3 Vitelliform macular dystrophy Bestrophin 1 BEST1 Vitreoretinochoroidopathy Bestrophin 1 BEST1

TABLE 6 Secreted Proteins Uniprot ID Protein Name Gene Name A1E959 Odontogenic ameloblast-associated protein ODAM A1KZ92 Peroxidasin-like protein PXDNL A1L453 Serine protease 38 PRSS38 A1L4H1 Soluble scavenger receptor cysteine-rich SSC5D domain-containing protein SSC5D A2RUU4 Colipase-like protein 1 CLPSL1 A2VDF0 Fucose mutarotase FUOM A2VEC9 SCO-spondin SSPO A3KMH1 von Willebrand factor A domain-containing VWA8 protein 8 A4D0S4 Laminin subunit beta-4 LAMB4 A4D1T9 Probable inactive serine protease 37 PRSS37 A5D8T8 C-type lectin domain family 18 member A CLEC18A A6NC86 phospholipase A2 inhibitor and Ly6/PLAUR PINLYP domain-containing protein A6NCI4 von Willebrand factor A domain-containing VWA3A protein 3A A6ND01 Probable folate receptor delta FOLR4 A6NDD2 Beta-defensin 108B-like A6NE02 BTB/POZ domain-containing protein 17 BTBD17 A6NEF6 Growth hormone 1 GH1 A6NF02 NPIP-like protein LOC730153 A6NFB4 HCG1749481, isoform CRA_k CSH1 A6NFZ4 Protein FAM24A FAM24A A6NG13 Glycosyltransferase 54 domain-containing protein A6NGN9 IgLON family member 5 IGLON5 A6NHN0 Otolin-1 OTOL1 A6NHN6 Nuclear pore complex-interacting protein-like 2 NPIPL2 A6NI73 Leukocyte immunoglobulin-like receptor LILRA5 subfamily A member 5 A6NIT4 Chorionic somatomammotropin hormone 2 CSH2 isoform 2 A6NJ69 IgA-inducing protein homolog IGIP A6NKQ9 Choriogonadotropin subunit beta variant 1 CGB1 A6NMZ7 Collagen alpha-6(VI) chain COL6A6 A6NNS2 Dehydrogenase/reductase SDR family member DHRS7C 7C A6XGL2 Insulin A chain INS A8K0G1 Protein Wnt WNT7B A8K2U0 Alpha-2-macroglobulin-like protein 1 A2ML1 A8K7I4 Calcium-activated chloride channel regulator 1 CLCA1 A8MTL9 Serpin-like protein HMSD HMSD A8MV23 Serpin E3 SERPINE3 A8MZH6 Oocyte-secreted protein 1 homolog OOSP1 A8TX70 Collagen alpha-5(VI) chain COL6A5 B0ZBE8 Natriuretic peptide NPPA B1A4G9 Somatotropin GH1 B1A4H2 HCG1749481, isoform CRA_d CSH1 B1A4H9 Chorionic somatomammotropin hormone CSH2 B1AJZ6 Protein Wnt WNT4 B1AKI9 Isthmin-1 ISM1 B2RNN3 Complement C1q and tumor necrosis factor- C1QTNF9B related protein 9B B2RUY7 von Willebrand factor C domain-containing VWC2L protein 2-like B3GLJ2 Prostate and testis expressed protein 3 PATE3 B4DI03 SEC11-like 3 (S. cerevisiae), isoform CRA_a SEC11L3 B4DJF9 Protein Wnt WNT4 B4DUL4 SEC11-like 1 (S. cerevisiae), isoform CRA_d SEC11L1 B5MCC8 Protein Wnt WNT10B B8A595 Protein Wnt WNT7B B8A597 Protein Wnt WNT7B B8A598 Protein Wnt WNT7B B9A064 Immunoglobulin lambda-like polypeptide 5 IGLL5 C9J3H3 Protein Wnt WNT10B C9J8I8 Protein Wnt WNT5A C9JAF2 Insulin-like growth factor II Ala-25 Del IGF2 C9JCI2 Protein Wnt WNT10B C9JL84 HERV-H LTR-associating protein 1 HHLA1 C9JNR5 Insulin A chain INS C9JUI2 Protein Wnt WNT2 D6RF47 Protein Wnt WNT8A D6RF94 Protein Wnt WNT8A E2RYF7 Protein PBMUCL2 HCG22 E5RFR1 PENK(114-133) PENK E7EML9 Serine protease 44 PRSS44 E7EPC3 Protein Wnt WNT9B E7EVP0 Nociceptin PNOC E9PD02 Insulin-like growth factor I IGF1 E9PH60 Protein Wnt WNT16 E9PJL6 Protein Wnt WNT11 F5GYM2 Protein Wnt WNT5B F5H034 Protein Wnt WNT5B F5H364 Protein Wnt WNT5B F5H7Q6 Protein Wnt WNT5B F8WCM5 Protein INS-IGF2 INS-IGF2 F8WDR1 Protein Wnt WNT2 H0Y663 Protein Wnt WNT4 H0YK72 Signal peptidase complex catalytic subunit SEC11A SEC11A H0YK83 Signal peptidase complex catalytic subunit SEC11A SEC11A H0YM39 Chorionic somatomammotropin hormone CSH2 H0YMT7 Chorionic somatomammotropin hormone CSH1 H0YN17 Chorionic somatomammotropin hormone CSH2 H0YNA5 Signal peptidase complex catalytic subunit SEC11A SEC11A H0YNG3 Signal peptidase complex catalytic subunit SEC11A SEC11A H0YNX5 Signal peptidase complex catalytic subunit SEC11A SEC11A H7BZB8 Protein Wnt WNT10A H9KV56 Choriogonadotropin subunit beta variant 2 CGB2 I3L0L8 Protein Wnt WNT9B J3KNZ1 Choriogonadotropin subunit beta variant 1 CGB1 J3KP00 Choriogonadotropin subunit beta CGB7 J3QT02 Choriogonadotropin subunit beta variant 1 CGB1 O00175 C-C motif chemokine 24 CCL24 O00182 Galectin-9 LGALS9 O00187 Mannan-binding lectin serine protease 2 MASP2 O00230 Cortistatin CORT O00253 Agouti-related protein AGRP O00270 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid GPR31 receptor O00292 Left-right determination factor 2 LEFTY2 O00294 Tubby-related protein 1 TULP1 O00295 Tubby-related protein 2 TULP2 O00300 Tumor necrosis factor receptor superfamily TNFRSF11B member 11B O00339 Matrilin-2 MATN2 O00391 Sulfhydryl oxidase 1 QSOX1 O00468 Agrin AGRN O00515 Ladinin-1 LAD1 O00533 Processed neural cell adhesion molecule L1-like CHL1 protein O00584 Ribonuclease T2 RNASET2 O00585 C-C motif chemokine 21 CCL21 O00602 Ficolin-1 FCN1 O00622 Protein CYR61 CYR61 O00626 MDC(5-69) CCL22 O00634 Netrin-3 NTN3 O00744 Protein Wnt-10b WNT10B O00755 Protein Wnt-7a WNT7A O14498 Immunoglobulin superfamily containing ISLR leucine-rich repeat protein O14511 Pro-neuregulin-2, membrane-bound isoform NRG2 O14594 Neurocan core protein NCAN O14625 C-X-C motif chemokine 11 CXCL11 O14638 Ectonucleotide pyrophosphatase/ ENPP3 phosphodiesterase family member 3 O14656 Torsin-1A TOR1A O14657 Torsin-1B TOR1B O14786 Neuropilin-1 NRP1 O14788 Tumor necrosis factor ligand superfamily TNFSF11 member 11, membrane form O14791 Apolipoprotein L1 APOL1 O14793 Growth/differentiation factor 8 MSTN O14904 Protein Wnt-9a WNT9A O14905 Protein Wnt-9b WNT9B O14944 Proepiregulin EREG O14960 Leukocyte cell-derived chemotaxin-2 LECT2 O15018 Processed PDZ domain-containing protein 2 PDZD2 O15041 Semaphorin-3E SEMA3E O15072 A disintegrin and metalloproteinase with ADAMTS3 thrombospondin motifs 3 O15123 Angiopoietin-2 ANGPT2 O15130 Neuropeptide FF NPFF O15197 Ephrin type-B receptor 6 EPHB6 O15204 ADAM DEC1 ADAMDEC1 O15230 Laminin subunit alpha-5 LAMA5 O15232 Matrilin-3 MATN3 O15240 Neuroendocrine regulatory peptide-1 VGF O15263 Beta-defensin 4A DEFB4A O15335 Chondroadherin CHAD O15393 Transmembrane protease serine 2 catalytic TMPRSS2 chain O15444 C-C motif chemokine 25 CCL25 O15467 C-C motif chemokine 16 CCL16 O15496 Group 10 secretory phospholipase A2 PLA2G10 O15520 Fibroblast growth factor 10 FGF10 O15537 Retinoschisin RS1 O43157 Plexin-B1 PLXNB1 O43184 Disintegrin and metalloproteinase domain- ADAM12 containing protein 12 O43240 Kallikrein-10 KLK10 O43278 Kunitz-type protease inhibitor 1 SPINT1 O43320 Fibroblast growth factor 16 FGF16 O43323 Desert hedgehog protein C-product DHH O43405 Cochlin COCH O43508 Tumor necrosis factor ligand superfamily TNFSF12 member 12, membrane form O43555 Progonadoliberin-2 GNRH2 O43557 Tumor necrosis factor ligand superfamily TNFSF14 member 14, soluble form O43692 Peptidase inhibitor 15 PI15 O43699 Sialic acid-binding Ig-like lectin 6 SIGLEC6 O43820 Hyaluronidase-3 HYAL3 O43827 Angiopoietin-related protein 7 ANGPTL7 O43852 Calumenin CALU O43854 EGF-like repeat and discoidin I-like domain- EDIL3 containing protein 3 O43866 CD5 antigen-like CD5L O43897 Tolloid-like protein 1 TLL1 O43915 Vascular endothelial growth factor D FIGF O43927 C-X-C motif chemokine 13 CXCL13 O60218 Aldo-keto reductase family 1 member B10 AKR1B10 O60235 Transmembrane protease serine 11D TMPRSS11D O60258 Fibroblast growth factor 17 FGF17 O60259 Kallikrein-8 KLK8 O60383 Growth/differentiation factor 9 GDF9 O60469 Down syndrome cell adhesion molecule DSCAM O60542 Persephin PSPN O60565 Gremlin-1 GREM1 O60575 Serine protease inhibitor Kazal-type 4 SPINK4 O60676 Cystatin-8 CST8 O60687 Sushi repeat-containing protein SRPX2 SRPX2 O60844 Zymogen granule membrane protein 16 ZG16 O60882 Matrix metalloproteinase-20 MMP20 O60938 Keratocan KERA O75015 Low affinity immunoglobulin gamma Fc region FCGR3B receptor III-B O75077 Disintegrin and metalloproteinase domain- ADAM23 containing protein 23 O75093 Slit homolog 1 protein SLIT1 O75094 Slit homolog 3 protein SLIT3 O75095 Multiple epidermal growth factor-like domains MEGF6 protein 6 O75173 A disintegrin and metalloproteinase with ADAMTS4 thrombospondin motifs 4 O75200 Nuclear pore complex-interacting protein-like 1 NPIPL1 O75339 Cartilage intermediate layer protein 1 C1 CILP O75354 Ectonucleoside triphosphate ENTPD6 diphosphohydrolase 6 O75386 Tubby-related protein 3 TULP3 O75398 Deformed epidermal autoregulatory factor 1 DEAF1 homolog O75443 Alpha-tectorin TECTA O75445 Usherin USH2A O75462 Cytokine receptor-like factor 1 CRLF1 O75487 Glypican-4 GPC4 O75493 Carbonic anhydrase-related protein 11 CA11 O75594 Peptidoglycan recognition protein 1 PGLYRP1 O75596 C-type lectin domain family 3 member A CLEC3A O75610 Left-right determination factor 1 LEFTY1 O75629 Protein CREG1 CREG1 O75636 Ficolin-3 FCN3 O75711 Scrapie-responsive protein 1 SCRG1 O75715 Epididymal secretory glutathione peroxidase GPX5 O75718 Cartilage-associated protein CRTAP O75829 Chondrosurfactant protein LECT1 O75830 Serpin I2 SERPINI2 O75882 Attractin ATRN O75888 Tumor necrosis factor ligand superfamily TNFSF13 member 13 O75900 Matrix metalloproteinase-23 MMP23A O75951 Lysozyme-like protein 6 LYZL6 O75973 C1q-related factor C1QL1 O76038 Secretagogin SCGN O76061 Stanniocalcin-2 STC2 O76076 WNT1-inducible-signaling pathway protein 2 WISP2 O76093 Fibroblast growth factor 18 FGF18 O76096 Cystatin-F CST7 O94769 Extracellular matrix protein 2 ECM2 O94813 Slit homolog 2 protein C-product SLIT2 O94907 Dickkopf-related protein 1 DKK1 O94919 Endonuclease domain-containing 1 protein ENDOD1 O94964 N-terminal form SOGA1 O95025 Semaphorin-3D SEMA3D O95084 Serine protease 23 PRSS23 O95150 Tumor necrosis factor ligand superfamily TNFSF15 member 15 O95156 Neurexophilin-2 NXPH2 O95157 Neurexophilin-3 NXPH3 O95158 Neurexophilin-4 NXPH4 O95388 WNT1-inducible-signaling pathway protein 1 WISP1 O95389 WNT1-inducible-signaling pathway protein 3 WISP3 O95390 Growth/differentiation factor 11 GDF11 O95393 Bone morphogenetic protein 10 BMP10 O95399 Urotensin-2 UTS2 O95407 Tumor necrosis factor receptor superfamily TNFRSF6B member 6B O95428 Papilin PAPLN O95445 Apolipoprotein M APOM O95450 A disintegrin and metalloproteinase with ADAMTS2 thrombospondin motifs 2 O95460 Matrilin-4 MATN4 O95467 LHAL tetrapeptide GNAS O95631 Netrin-1 NTN1 O95633 Follistatin-related protein 3 FSTL3 O95711 Lymphocyte antigen 86 LY86 O95715 C-X-C motif chemokine 14 CXCL14 O95750 Fibroblast growth factor 19 FGF19 O95760 Interleukin-33 IL33 O95813 Cerberus CER1 O95841 Angiopoietin-related protein 1 ANGPTL1 O95897 Noelin-2 OLFM2 O95925 Eppin EPPIN O95965 Integrin beta-like protein 1 ITGBL1 O95967 EGF-containing fibulin-like extracellular matrix EFEMP2 protein 2 O95968 Secretoglobin family 1D member 1 SCGB1D1 O95969 Secretoglobin family 1D member 2 SCGB1D2 O95970 Leucine-rich glioma-inactivated protein 1 LGI1 O95972 Bone morphogenetic protein 15 BMP15 O95994 Anterior gradient protein 2 homolog AGR2 O95998 Interleukin-18-binding protein IL18BP O96009 Napsin-A NAPSA O96014 Protein Wnt-11 WNT11 P00450 Ceruloplasmin CP P00451 Factor VIIIa light chain F8 P00488 Coagulation factor XIII A chain F13A1 P00533 Epidermal growth factor receptor EGFR P00709 Alpha-lactalbumin LALBA P00734 Prothrombin F2 P00738 Haptoglobin beta chain HP P00739 Haptoglobin-related protein HPR P00740 Coagulation factor IXa heavy chain F9 P00742 Factor X heavy chain F10 P00746 Complement factor D CFD P00747 Plasmin light chain B PLG P00748 Coagulation factor XIIa light chain F12 P00749 Urokinase-type plasminogen activator long PLAU chain A P00750 Tissue-type plasminogen activator PLAT P00751 Complement factor B Ba fragment CFB P00797 Renin REN P00973 2′-5′-oligoadenylate synthase 1 OAS1 P00995 Pancreatic secretory trypsin inhibitor SPINK1 P01008 Antithrombin-III SERPINC1 P01009 Alpha-1-antitrypsin SERPINA1 P01011 Alpha-1-antichymotrypsin His-Pro-less SERPINA3 P01019 Angiotensin-1 AGT P01023 Alpha-2-macroglobulin A2M P01024 Acylation stimulating protein C3 P01031 Complement C5 beta chain C5 P01033 Metalloproteinase inhibitor 1 TIMP1 P01034 Cystatin-C CST3 P01036 Cystatin-S CST4 P01037 Cystatin-SN CST1 P01042 Kininogen-1 light chain KNG1 P01127 Platelet-derived growth factor subunit B PDGFB P01135 Transforming growth factor alpha TGFA P01137 Transforming growth factor beta-1 TGFB1 P01138 Beta-nerve growth factor NGF P01148 Gonadoliberin-1 GNRH1 P01160 Atrial natriuretic factor NPPA P01178 Oxytocin OXT P01185 Vasopressin-neurophysin 2-copeptin AVP P01189 Corticotropin POMC P01210 PENK(237-258) PENK P01213 Alpha-neoendorphin PDYN P01215 Glycoprotein hormones alpha chain CGA P01222 Thyrotropin subunit beta TSHB P01225 Follitropin subunit beta FSHB P01229 Lutropin subunit beta LHB P01233 Choriogonadotropin subunit beta CGB8 P01236 Prolactin PRL P01241 Somatotropin GH1 P01242 Growth hormone variant GH2 P01243 Chorionic somatomammotropin hormone CSH2 P01258 Katacalcin CALCA P01266 Thyroglobulin TG P01270 Parathyroid hormone PTH P01275 Glucagon GCG P01282 Intestinal peptide PHM-27 VIP P01286 Somatoliberin GHRH P01298 Pancreatic prohormone PPY P01303 C-flanking peptide of NPY NPY P01308 Insulin INS P01344 Insulin-like growth factor II IGF2 P01350 Big gastrin GAST P01374 Lymphotoxin-alpha LTA P01375 C-domain 1 TNF P01562 Interferon alpha-1/13 IFNA1 P01563 Interferon alpha-2 IFNA2 P01566 Interferon alpha-10 IFNA10 P01567 Interferon alpha-7 IFNA7 P01568 Interferon alpha-21 IFNA21 P01569 Interferon alpha-5 IFNA5 P01570 Interferon alpha-14 IFNA14 P01571 Interferon alpha-17 IFNA17 P01574 Interferon beta IFNB1 P01579 Interferon gamma IFNG P01583 Interleukin-1 alpha IL1A P01584 Interleukin-1 beta IL1B P01588 Erythropoietin EPO P01591 Immunoglobulin J chain IGJ P01732 T-cell surface glycoprotein CD8 alpha chain CD8A P01833 Polymeric immunoglobulin receptor PIGR P01857 Ig gamma-1 chain C region IGHG1 P01859 Ig gamma-2 chain C region IGHG2 P01860 Ig gamma-3 chain C region IGHG3 P01861 Ig gamma-4 chain C region IGHG4 P01871 Ig mu chain C region IGHM P01880 Ig delta chain C region IGHD P02452 Collagen alpha-1(I) chain COL1A1 P02458 Chondrocalcin COL2A1 P02461 Collagen alpha-1(III) chain COL3A1 P02462 Collagen alpha-1(IV) chain COL4A1 P02647 Apolipoprotein A-I APOA1 P02649 Apolipoprotein E APOE P02652 Apolipoprotein A-II APOA2 P02654 Apolipoprotein C-I APOC1 P02655 Apolipoprotein C-II APOC2 P02656 Apolipoprotein C-III APOC3 P02671 Fibrinogen alpha chain FGA P02675 Fibrinopeptide B FGB P02679 Fibrinogen gamma chain FGG P02741 C-reactive protein CRP P02743 Serum amyloid P-component(1-203) APCS P02745 Complement C1q subcomponent subunit A C1QA P02746 Complement C1q subcomponent subunit B C1QB P02747 Complement C1q subcomponent subunit C C1QC P02748 Complement component C9b C9 P02749 Beta-2-glycoprotein 1 APOH P02750 Leucine-rich alpha-2-glycoprotein LRG1 P02751 Ugl-Y2 FN1 P02753 Retinol-binding protein 4 RBP4 P02760 Trypstatin AMBP P02763 Alpha-1-acid glycoprotein 1 ORM1 P02765 Alpha-2-HS-glycoprotein chain A AHSG P02766 Transthyretin TTR P02768 Serum albumin ALB P02771 Alpha-fetoprotein AFP P02774 Vitamin D-binding protein GC P02775 Connective tissue-activating peptide III PPBP P02776 Platelet factor 4 PF4 P02778 CXCL10(1-73) CXCL10 P02786 Transferrin receptor protein 1 TFRC P02787 Serotransferrin TF P02788 Lactoferroxin-C LTF P02790 Hemopexin HPX P02808 Statherin STATH P02810 Salivary acidic proline-rich phosphoprotein 1/2 PRH2 P02812 Basic salivary proline-rich protein 2 PRB2 P02814 Peptide D1A SMR3B P02818 Osteocalcin BGLAP P03950 Angiogenin ANG P03951 Coagulation factor XIa heavy chain F11 P03952 Plasma kallikrein KLKB1 P03956 27 kDa interstitial collagenase MMP1 P03971 Muellerian-inhibiting factor AMH P03973 Antileukoproteinase SLPI P04003 C4b-binding protein alpha chain C4BPA P04004 Somatomedin-B VTN P04054 Phospholipase A2 PLA2G1B P04085 Platelet-derived growth factor subunit A PDGFA P04090 Relaxin A chain RLN2 P04114 Apolipoprotein B-100 APOB P04118 Colipase CLPS P04141 Granulocyte-macrophage colony-stimulating CSF2 factor P04155 Trefoil factor 1 TFF1 P04180 Phosphatidylcholine-sterol acyltransferase LCAT P04196 Histidine-rich glycoprotein HRG P04217 Alpha-1B-glycoprotein A1BG P04275 von Willebrand antigen 2 VWF P04278 Sex hormone-binding globulin SHBG P04279 Alpha-inhibin-31 SEMG1 P04280 Basic salivary proline-rich protein 1 PRB1 P04628 Proto-oncogene Wnt-1 WNT1 P04745 Alpha-amylase 1 AMY1A P04746 Pancreatic alpha-amylase AMY2A P04808 Prorelaxin H1 RLN1 P05000 Interferon omega-1 IFNW1 P05013 Interferon alpha-6 IFNA6 P05014 Interferon alpha-4 IFNA4 P05015 Interferon alpha-16 IFNA16 P05019 Insulin-like growth factor I IGF1 P05060 GAWK peptide CHGB P05090 Apolipoprotein D APOD P05109 Protein S100-A8 S100A8 P05111 Inhibin alpha chain INHA P05112 Interleukin-4 IL4 P05113 Interleukin-5 IL5 P05120 Plasminogen activator inhibitor 2 SERPINB2 P05121 Plasminogen activator inhibitor 1 SERPINE1 P05154 Plasma serine protease inhibitor SERPINA5 P05155 Plasma protease C1 inhibitor SERPING1 P05156 Complement factor I heavy chain CFI P05160 Coagulation factor XIII B chain F13B P05161 Ubiquitin-like protein ISG15 ISG15 P05230 Fibroblast growth factor 1 FGF1 P05231 Interleukin-6 IL6 P05305 Big endothelin-1 EDN1 P05408 C-terminal peptide SCG5 P05451 Lithostathine-1-alpha REG1A P05452 Tetranectin CLEC3B P05543 Thyroxine-binding globulin SERPINA7 P05814 Beta-casein CSN2 P05997 Collagen alpha-2(V) chain COL5A2 P06276 Cholinesterase BCHE P06307 Cholecystokinin-12 CCK P06396 Gelsolin GSN P06681 Complement C2 C2 P06702 Protein S100-A9 S100A9 P06727 Apolipoprotein A-IV APOA4 P06734 Low affinity immunoglobulin epsilon Fc FCER2 receptor soluble form P06744 Glucose-6-phosphate isomerase GPI P06850 Corticoliberin CRH P06858 Lipoprotein lipase LPL P06881 Calcitonin gene-related peptide 1 CALCA P07093 Glia-derived nexin SERPINE2 P07098 Gastric triacylglycerol lipase LIPF P07225 Vitamin K-dependent protein S PROS1 P07237 Protein disulfide-isomerase P4HB P07288 Prostate-specific antigen KLK3 P07306 Asialoglycoprotein receptor 1 ASGR1 P07355 Annexin A2 ANXA2 P07357 Complement component C8 alpha chain C8A P07358 Complement component C8 beta chain C8B P07360 Complement component C8 gamma chain C8G P07477 Alpha-trypsin chain 2 PRSS1 P07478 Trypsin-2 PRSS2 P07492 Neuromedin-C GRP P07498 Kappa-casein CSN3 P07585 Decorin DCN P07911 Uromodulin UMOD P07942 Laminin subunit beta-1 LAMB1 P07988 Pulmonary surfactant-associated protein B SFTPB P07998 Ribonuclease pancreatic RNASE1 P08118 Beta-microseminoprotein MSMB P08123 Collagen alpha-2(I) chain COL1A2 P08185 Corticosteroid-binding globulin SERPINA6 P08217 Chymotrypsin-like elastase family member 2A CELA2A P08218 Chymotrypsin-like elastase family member 2B CELA2B P08253 72 kDa type IV collagenase MMP2 P08254 Stromelysin-1 MMP3 P08294 Extracellular superoxide dismutase [Cu—Zn] SOD3 P08476 Inhibin beta A chain INHBA P08493 Matrix Gla protein MGP P08572 Collagen alpha-2(IV) chain COL4A2 P08581 Hepatocyte growth factor receptor MET P08603 Complement factor H CFH P08620 Fibroblast growth factor 4 FGF4 P08637 Low affinity immunoglobulin gamma Fc region FCGR3A receptor III-A P08697 Alpha-2-antiplasmin SERPINF2 P08700 Interleukin-3 IL3 P08709 Coagulation factor VII F7 P08833 Insulin-like growth factor-binding protein 1 IGFBP1 P08887 Interleukin-6 receptor subunit alpha IL6R P08949 Neuromedin-B-32 NMB P08F94 Fibrocystin PKHD1 P09038 Fibroblast growth factor 2 FGF2 P09228 Cystatin-SA CST2 P09237 Matrilysin MMP7 P09238 Stromelysin-2 MMP10 P09341 Growth-regulated alpha protein CXCL1 P09382 Galectin-1 LGALS1 P09466 Glycodelin PAEP P09486 SPARC SPARC P09529 Inhibin beta B chain INHBB P09544 Protein Wnt-2 WNT2 P09603 Processed macrophage colony-stimulating CSF1 factor 1 P09681 Gastric inhibitory polypeptide GIP P09683 Secretin SCT P09919 Granulocyte colony-stimulating factor CSF3 P0C091 FRAS1-related extracellular matrix protein 3 FREM3 P0C0L4 C4d-A C4A P0C0L5 Complement C4-B alpha chain C4B P0C0P6 Neuropeptide S NPS P0C7L1 Serine protease inhibitor Kazal-type 8 SPINK8 P0C862 Complement C1q and tumor necrosis factor- C1QTNF9 related protein 9A P0C8F1 Prostate and testis expressed protein 4 PATE4 P0CG01 Gastrokine-3 GKN3P P0CG36 Cryptic family protein 1B CFC1B P0CG37 Cryptic protein CFC1 P0CJ68 Humanin-like protein 1 MTRNR2L1 P0CJ69 Humanin-like protein 2 MTRNR2L2 P0CJ70 Humanin-like protein 3 MTRNR2L3 P0CJ71 Humanin-like protein 4 MTRNR2L4 P0CJ72 Humanin-like protein 5 MTRNR2L5 P0CJ73 Humanin-like protein 6 MTRNR2L6 P0CJ74 Humanin-like protein 7 MTRNR2L7 P0CJ75 Humanin-like protein 8 MTRNR2L8 P0CJ76 Humanin-like protein 9 MTRNR2L9 P0CJ77 Humanin-like protein 10 MTRNR2L10 P0DJD7 Pepsin A-4 PGA4 P0DJD8 Pepsin A-3 PGA3 P0DJD9 Pepsin A-5 PGA5 P0DJI8 Amyloid protein A SAA1 P0DJI9 Serum amyloid A-2 protein SAA2 P10082 Peptide YY(3-36) PYY P10092 Calcitonin gene-related peptide 2 CALCB P10124 Serglycin SRGN P10145 MDNCF-a IL8 P10147 MIP-1-alpha(4-69) CCL3 P10163 Peptide P-D PRB4 P10451 Osteopontin SPP1 P10599 Thioredoxin TXN P10600 Transforming growth factor beta-3 TGFB3 P10643 Complement component C7 C7 P10645 Vasostatin-2 CHGA P10646 Tissue factor pathway inhibitor TFPI P10720 Platelet factor 4 variant(4-74) PF4V1 P10745 Retinol-binding protein 3 RBP3 P10767 Fibroblast growth factor 6 FGF6 P10909 Clusterin alpha chain CLU P10912 Growth hormone receptor GHR P10915 Hyaluronan and proteoglycan link protein 1 HAPLN1 P10966 T-cell surface glycoprotein CD8 beta chain CD8B P10997 Islet amyloid polypeptide IAPP P11047 Laminin subunit gamma-1 LAMC1 P11150 Hepatic triacylglycerol lipase LIPC P11226 Mannose-binding protein C MBL2 P11464 Pregnancy-specific beta-1-glycoprotein 1 PSG1 P11465 Pregnancy-specific beta-1-glycoprotein 2 PSG2 P11487 Fibroblast growth factor 3 FGF3 P11597 Cholesteryl ester transfer protein CETP P11684 Uteroglobin SCGB1A1 P11686 Pulmonary surfactant-associated protein C SFTPC P12034 Fibroblast growth factor 5 FGF5 P12107 Collagen alpha-1(XI) chain COL11A1 P12109 Collagen alpha-1(VI) chain COL6A1 P12110 Collagen alpha-2(VI) chain COL6A2 P12111 Collagen alpha-3(VI) chain COL6A3 P12259 Coagulation factor V F5 P12272 PTHrP[1-36] PTHLH P12273 Prolactin-inducible protein PIP P12544 Granzyme A GZMA P12643 Bone morphogenetic protein 2 BMP2 P12644 Bone morphogenetic protein 4 BMP4 P12645 Bone morphogenetic protein 3 BMP3 P12724 Eosinophil cationic protein RNASE3 P12821 Angiotensin-converting enzyme, soluble form ACE P12838 Neutrophil defensin 4 DEFA4 P12872 Motilin MLN P13232 Interleukin-7 IL7 P13236 C-C motif chemokine 4 CCL4 P13284 Gamma-interferon-inducible lysosomal thiol IFI30 reductase P13500 C-C motif chemokine 2 CCL2 P13501 C-C motif chemokine 5 CCL5 P13521 Secretogranin-2 SCG2 P13591 Neural cell adhesion molecule 1 NCAM1 P13611 Versican core protein VCAN P13671 Complement component C6 C6 P13688 Carcinoembryonic antigen-related cell CEACAM1 adhesion molecule 1 P13725 Oncostatin-M OSM P13726 Tissue factor F3 P13727 Eosinophil granule major basic protein PRG2 P13942 Collagen alpha-2(XI) chain COL11A2 P13987 CD59 glycoprotein CD59 P14138 Endothelin-3 EDN3 P14174 Macrophage migration inhibitory factor MIF P14207 Folate receptor beta FOLR2 P14222 Perforin-1 PRF1 P14543 Nidogen-1 NID1 P14555 Phospholipase A2, membrane associated PLA2G2A P14625 Endoplasmin HSP90B1 P14735 Insulin-degrading enzyme IDE P14778 Interleukin-1 receptor type 1, soluble form IL1R1 P14780 82 kDa matrix metalloproteinase-9 MMP9 P15018 Leukemia inhibitory factor LIF P15085 Carboxypeptidase A1 CPA1 P15086 Carboxypeptidase B CPB1 P15151 Poliovirus receptor PVR P15169 Carboxypeptidase N catalytic chain CPN1 P15248 Interleukin-9 IL9 P15291 N-acetyllactosamine synthase B4GALT1 P15309 PAPf39 ACPP P15328 Folate receptor alpha FOLR1 P15374 Ubiquitin carboxyl-terminal hydrolase isozyme UCHL3 L3 P15502 Elastin ELN P15509 Granulocyte-macrophage colony-stimulating CSF2RA factor receptor subunit alpha P15515 Histatin-1 HTN1 P15516 His3-(31-51)-peptide HTN3 P15692 Vascular endothelial growth factor A VEGFA P15814 Immunoglobulin lambda-like polypeptide 1 IGLL1 P15907 Beta-galactoside alpha-2,6-sialyltransferase 1 ST6GAL1 P15941 Mucin-1 subunit beta MUC1 P16035 Metalloproteinase inhibitor 2 TIMP2 P16112 Aggrecan core protein 2 ACAN P16233 Pancreatic triacylglycerol lipase PNLIP P16442 Histo-blood group ABO system transferase ABO P16471 Prolactin receptor PRLR P16562 Cysteine-rich secretory protein 2 CRISP2 P16619 C-C motif chemokine 3-like 1 CCL3L1 P16860 BNP(3-29) NPPB P16870 Carboxypeptidase E CPE P16871 Interleukin-7 receptor subunit alpha IL7R P17213 Bactericidal permeability-increasing protein BPI P17538 Chymotrypsinogen B CTRB1 P17931 Galectin-3 LGALS3 P17936 Insulin-like growth factor-binding protein 3 IGFBP3 P17948 Vascular endothelial growth factor receptor 1 FLT1 P18065 Insulin-like growth factor-binding protein 2 IGFBP2 P18075 Bone morphogenetic protein 7 BMP7 P18428 Lipopolysaccharide-binding protein LBP P18509 PACAP-related peptide ADCYAP1 P18510 Interleukin-1 receptor antagonist protein IL1RN P18827 Syndecan-1 SDC1 P19021 Peptidylglycine alpha-hydroxylating PAM monooxygenase P19235 Erythropoietin receptor EPOR P19438 Tumor necrosis factor-binding protein 1 TNFRSF1A P19652 Alpha-1-acid glycoprotein 2 ORM2 P19801 Amiloride-sensitive amine oxidase [copper- ABP1 containing] P19823 Inter-alpha-trypsin inhibitor heavy chain H2 ITIH2 P19827 Inter-alpha-trypsin inhibitor heavy chain H1 ITIH1 P19835 Bile salt-activated lipase CEL P19875 C-X-C motif chemokine 2 CXCL2 P19876 C-X-C motif chemokine 3 CXCL3 P19883 Follistatin FST P19957 Elafin PI3 P19961 Alpha-amylase 2B AMY2B P20061 Transcobalamin-1 TCN1 P20062 Transcobalamin-2 TCN2 P20142 Gastricsin PGC P20155 Serine protease inhibitor Kazal-type 2 SPINK2 P20231 Tryptase beta-2 TPSB2 P20333 Tumor necrosis factor receptor superfamily TNFRSF1B member 1B P20366 Substance P TAC1 P20382 Melanin-concentrating hormone PMCH P20396 Thyroliberin TRH P20742 Pregnancy zone protein PZP P20774 Mimecan OGN P20783 Neurotrophin-3 NTF3 P20800 Endothelin-2 EDN2 P20809 Interleukin-11 IL11 P20827 Ephrin-A1 EFNA1 P20849 Collagen alpha-1(IX) chain COL9A1 P20851 C4b-binding protein beta chain C4BPB P20908 Collagen alpha-1(V) chain COL5A1 P21128 Poly(U)-specific endoribonuclease ENDOU P21246 Pleiotrophin PTN P21583 Kit ligand KITLG P21741 Midkine MDK P21754 Zona pellucida sperm-binding protein 3 ZP3 P21781 Fibroblast growth factor 7 FGF7 P21802 Fibroblast growth factor receptor 2 FGFR2 P21810 Biglycan BGN P21815 Bone sialoprotein 2 IBSP P21860 Receptor tyrosine-protein kinase erbB-3 ERBB3 P21941 Cartilage matrix protein MATN1 P22003 Bone morphogenetic protein 5 BMP5 P22004 Bone morphogenetic protein 6 BMP6 P22079 Lactoperoxidase LPO P22105 Tenascin-X TNXB P22301 Interleukin-10 IL10 P22303 Acetylcholinesterase ACHE P22352 Glutathione peroxidase 3 GPX3 P22362 C-C motif chemokine 1 CCL1 P22455 Fibroblast growth factor receptor 4 FGFR4 P22466 Galanin message-associated peptide GAL P22692 Insulin-like growth factor-binding protein 4 IGFBP4 P22749 Granulysin GNLY P22792 Carboxypeptidase N subunit 2 CPN2 P22891 Vitamin K-dependent protein Z PROZ P22894 Neutrophil collagenase MMP8 P23142 Fibulin-1 FBLN1 P23280 Carbonic anhydrase 6 CA6 P23352 Anosmin-1 KAL1 P23435 Cerebellin-1 CBLN1 P23560 Brain-derived neurotrophic factor BDNF P23582 C-type natriuretic peptide NPPC P23946 Chymase CMA1 P24043 Laminin subunit alpha-2 LAMA2 P24071 Immunoglobulin alpha Fc receptor FCAR P24347 Stromelysin-3 MMP11 P24387 Corticotropin-releasing factor-binding protein CRHBP P24592 Insulin-like growth factor-binding protein 6 IGFBP6 P24593 Insulin-like growth factor-binding protein 5 IGFBP5 P24821 Tenascin TNC P24855 Deoxyribonuclease-1 DNASE1 P25067 Collagen alpha-2(VIII) chain COL8A2 P25311 Zinc-alpha-2-glycoprotein AZGP1 P25391 Laminin subunit alpha-1 LAMA1 P25445 Tumor necrosis factor receptor superfamily FAS member 6 P25940 Collagen alpha-3(V) chain COL5A3 P25942 Tumor necrosis factor receptor superfamily CD40 member 5 P26022 Pentraxin-related protein PTX3 PTX3 P26927 Hepatocyte growth factor-like protein beta MST1 chain P27169 Serum paraoxonase/arylesterase 1 PON1 P27352 Gastric intrinsic factor GIF P27487 Dipeptidyl peptidase 4 membrane form DPP4 P27539 Embryonic growth/differentiation factor 1 GDF1 P27658 Vastatin COL8A1 P27797 Calreticulin CALR P27918 Properdin CFP P28039 Acyloxyacyl hydrolase AOAH P28300 Protein-lysine 6-oxidase LOX P28325 Cystatin-D CST5 P28799 Granulin-1 GRN P29122 Proprotein convertase subtilisin/kexin type 6 PCSK6 P29279 Connective tissue growth factor CTGF P29320 Ephrin type-A receptor 3 EPHA3 P29400 Collagen alpha-5(IV) chain COL4A5 P29459 Interleukin-12 subunit alpha IL12A P29460 Interleukin-12 subunit beta IL12B P29508 Serpin B3 SERPINB3 P29622 Kallistatin SERPINA4 P29965 CD40 ligand, soluble form CD40LG P30990 Neurotensin/neuromedin N NTS P31025 Lipocalin-1 LCN1 P31151 Protein S100-A7 S100A7 P31371 Fibroblast growth factor 9 FGF9 P31431 Syndecan-4 SDC4 P31947 14-3-3 protein sigma SFN P32455 Interferon-induced guanylate-binding protein 1 GBP1 P32881 Interferon alpha-8 IFNA8 P34096 Ribonuclease 4 RNASE4 P34130 Neurotrophin-4 NTF4 P34820 Bone morphogenetic protein 8B BMP8B P35030 Trypsin-3 PRSS3 P35052 Secreted glypican-1 GPC1 P35070 Betacellulin BTC P35225 Interleukin-13 IL13 P35247 Pulmonary surfactant-associated protein D SFTPD P35318 ADM ADM P35542 Serum amyloid A-4 protein SAA4 P35555 Fibrillin-1 FBN1 P35556 Fibrillin-2 FBN2 P35625 Metalloproteinase inhibitor 3 TIMP3 P35858 Insulin-like growth factor-binding protein IGFALS complex acid labile subunit P35916 Vascular endothelial growth factor receptor 3 FLT4 P35968 Vascular endothelial growth factor receptor 2 KDR P36222 Chitinase-3-like protein 1 CHI3L1 P36952 Serpin B5 SERPINB5 P36955 Pigment epithelium-derived factor SERPINF1 P36980 Complement factor H-related protein 2 CFHR2 P39059 Collagen alpha-1(XV) chain COL15A1 P39060 Collagen alpha-1(XVIII) chain COL18A1 P39877 Calcium-dependent phospholipase A2 PLA2G5 P39900 Macrophage metalloelastase MMP12 P39905 Glial cell line-derived neurotrophic factor GDNF P40225 Thrombopoietin THPO P40967 M-alpha PMEL P41159 Leptin LEP P41221 Protein Wnt-5a WNT5A P41222 Prostaglandin-H2 D-isomerase PTGDS P41271 Neuroblastoma suppressor of tumorigenicity 1 NBL1 P41439 Folate receptor gamma FOLR3 P42127 Agouti-signaling protein ASIP P42702 Leukemia inhibitory factor receptor LIFR P42830 ENA-78(9-78) CXCL5 P43026 Growth/differentiation factor 5 GDF5 P43251 Biotinidase BTD P43652 Afamin AFM P45452 Collagenase 3 MMP13 P47710 Casoxin-D CSN1S1 P47929 Galectin-7 LGALS7B P47972 Neuronal pentraxin-2 NPTX2 P47989 Xanthine oxidase XDH P47992 Lymphotactin XCL1 P48023 Tumor necrosis factor ligand superfamily FASLG member 6, membrane form P48052 Carboxypeptidase A2 CPA2 P48061 Stromal cell-derived factor 1 CXCL12 P48304 Lithostathine-1-beta REG1B P48307 Tissue factor pathway inhibitor 2 TFPI2 P48357 Leptin receptor LEPR P48594 Serpin B4 SERPINB4 P48645 Neuromedin-U-25 NMU P48740 Mannan-binding lectin serine protease 1 MASP1 P48745 Protein NOV homolog NOV P48960 CD97 antigen subunit beta CD97 P49223 Kunitz-type protease inhibitor 3 SPINT3 P49747 Cartilage oligomeric matrix protein COMP P49763 Placenta growth factor PGF P49765 Vascular endothelial growth factor B VEGFB P49767 Vascular endothelial growth factor C VEGFC P49771 Fms-related tyrosine kinase 3 ligand FLT3LG P49862 Kallikrein-7 KLK7 P49863 Granzyme K GZMK P49908 Selenoprotein P SEPP1 P49913 Antibacterial protein FALL-39 CAMP P50607 Tubby protein homolog TUB P51124 Granzyme M GZMM P51512 Matrix metalloproteinase-16 MMP16 P51654 Glypican-3 GPC3 P51671 Eotaxin CCL11 P51884 Lumican LUM P51888 Prolargin PRELP P52798 Ephrin-A4 EFNA4 P52823 Stanniocalcin-1 STC1 P53420 Collagen alpha-4(IV) chain COL4A4 P53621 Coatomer subunit alpha COPA P54108 Cysteine-rich secretory protein 3 CRISP3 P54315 Pancreatic lipase-related protein 1 PNLIPRP1 P54317 Pancreatic lipase-related protein 2 PNLIPRP2 P54793 Arylsulfatase F ARSF P55000 Secreted Ly-6/uPAR-related protein 1 SLURP1 P55001 Microfibrillar-associated protein 2 MFAP2 P55056 Apolipoprotein C-IV APOC4 P55058 Phospholipid transfer protein PLTP P55075 Fibroblast growth factor 8 FGF8 P55081 Microfibrillar-associated protein 1 MFAP1 P55083 Microfibril-associated glycoprotein 4 MFAP4 P55107 Bone morphogenetic protein 3B GDF10 P55145 Mesencephalic astrocyte-derived neurotrophic MANF factor P55259 Pancreatic secretory granule membrane major GP2 glycoprotein GP2 P55268 Laminin subunit beta-2 LAMB2 P55773 CCL23(30-99) CCL23 P55774 C-C motif chemokine 18 CCL18 P55789 FAD-linked sulfhydryl oxidase ALR GFER P56703 Proto-oncogene Wnt-3 WNT3 P56704 Protein Wnt-3a WNT3A P56705 Protein Wnt-4 WNT4 P56706 Protein Wnt-7b WNT7B P56730 Neurotrypsin PRSS12 P56851 Epididymal secretory protein E3-beta EDDM3B P56975 Neuregulin-3 NRG3 P58062 Serine protease inhibitor Kazal-type 7 SPINK7 P58215 Lysyl oxidase homolog 3 LOXL3 P58294 Prokineticin-1 PROK1 P58335 Anthrax toxin receptor 2 ANTXR2 P58397 A disintegrin and metalloproteinase with ADAMTS12 thrombospondin motifs 12 P58417 Neurexophilin-1 NXPH1 P58499 Protein FAM3B FAM3B P59510 A disintegrin and metalloproteinase with ADAMTS20 thrombospondin motifs 20 P59665 Neutrophil defensin 1 DEFA1B P59666 Neutrophil defensin 3 DEFA3 P59796 Glutathione peroxidase 6 GPX6 P59826 BPI fold-containing family B member 3 BPIFB3 P59827 BPI fold-containing family B member 4 BPIFB4 P59861 Beta-defensin 131 DEFB131 P60022 Beta-defensin 1 DEFB1 P60153 Inactive ribonuclease-like protein 9 RNASE9 P60827 Complement C1q tumor necrosis factor-related C1QTNF8 protein 8 P60852 Zona pellucida sperm-binding protein 1 ZP1 P60985 Keratinocyte differentiation-associated protein KRTDAP P61109 Kidney androgen-regulated protein KAP P61278 Somatostatin-14 SST P61366 Osteocrin OSTN P61626 Lysozyme C LYZ P61769 Beta-2-microglobulin B2M P61812 Transforming growth factor beta-2 TGFB2 P61916 Epididymal secretory protein E1 NPC2 P62502 Epididymal-specific lipocalin-6 LCN6 P62937 Peptidyl-prolyl cis-trans isomerase A PPIA P67809 Nuclease-sensitive element-binding protein 1 YBX1 P67812 Signal peptidase complex catalytic subunit SEC11A SEC11A P78310 Coxsackievirus and adenovirus receptor CXADR P78333 Secreted glypican-5 GPC5 P78380 Oxidized low-density lipoprotein receptor 1 OLR1 P78423 Processed fractalkine CX3CL1 P78509 Reelin RELN P78556 CCL20(2-70) CCL20 P80075 MCP-2(6-76) CCL8 P80098 C-C motif chemokine 7 CCL7 P80108 Phosphatidylinositol-glycan-specific GPLD1 phospholipase D P80162 C-X-C motif chemokine 6 CXCL6 P80188 Neutrophil gelatinase-associated lipocalin LCN2 P80303 Nucleobindin-2 NUCB2 P80511 Calcitermin S100A12 P81172 Hepcidin-25 HAMP P81277 Prolactin-releasing peptide PRLH P81534 Beta-defensin 103 DEFB103A P81605 Dermcidin DCD P82279 Protein crumbs homolog 1 CRB1 P82987 ADAMTS-like protein 3 ADAMTSL3 P83105 Serine protease HTRA4 HTRA4 P83110 Serine protease HTRA3 HTRA3 P83859 Orexigenic neuropeptide QRFP QRFP P98088 Mucin-5AC MUC5AC P98095 Fibulin-2 FBLN2 P98160 Basement membrane-specific heparan sulfate HSPG2 proteoglycan core protein P98173 Protein FAM3A FAM3A Q00604 Norrin NDP Q00796 Sorbitol dehydrogenase SORD Q00887 Pregnancy-specific beta-1-glycoprotein 9 PSG9 Q00888 Pregnancy-specific beta-1-glycoprotein 4 PSG4 Q00889 Pregnancy-specific beta-1-glycoprotein 6 PSG6 Q01523 HD5(56-94) DEFA5 Q01524 Defensin-6 DEFA6 Q01955 Collagen alpha-3(IV) chain COL4A3 Q02297 Pro-neuregulin-1, membrane-bound isoform NRG1 Q02325 Plasminogen-like protein B PLGLB1 Q02383 Semenogelin-2 SEMG2 Q02388 Collagen alpha-1(VII) chain COL7A1 Q02505 Mucin-3A MUC3A Q02509 Otoconin-90 OC90 Q02747 Guanylin GUCA2A Q02763 Angiopoietin-1 receptor TEK Q02817 Mucin-2 MUC2 Q02985 Complement factor H-related protein 3 CFHR3 Q03167 Transforming growth factor beta receptor type TGFBR3 3 Q03403 Trefoil factor 2 TFF2 Q03405 Urokinase plasminogen activator surface PLAUR receptor Q03591 Complement factor H-related protein 1 CFHR1 Q03692 Collagen alpha-1(X) chain COL10A1 Q04118 Basic salivary proline-rich protein 3 PRB3 Q04756 Hepatocyte growth factor activator short chain HGFAC Q04900 Sialomucin core protein 24 CD164 Q05315 Eosinophil lysophospholipase CLC Q05707 Collagen alpha-1(XIV) chain COL14A1 Q05996 Processed zona pellucida sperm-binding ZP2 protein 2 Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 ITIH3 Q06141 Regenerating islet-derived protein 3-alpha REG3A Q06828 Fibromodulin FMOD Q07092 Collagen alpha-1(XVI) chain COL16A1 Q07325 C-X-C motif chemokine 9 CXCL9 Q07507 Dermatopontin DPT Q075Z2 Binder of sperm protein homolog 1 BSPH1 Q07654 Trefoil factor 3 TFF3 Q07699 Sodium channel subunit beta-1 SCN1B Q08345 Epithelial discoidin domain-containing receptor DDR1 1 Q08380 Galectin-3-binding protein LGALS3BP Q08397 Lysyl oxidase homolog 1 LOXL1 Q08431 Lactadherin MFGE8 Q08629 Testican-1 SPOCK1 Q08648 Sperm-associated antigen 11B SPAG11B Q08830 Fibrinogen-like protein 1 FGL1 Q10471 Polypeptide N-acetylgalactosaminyltransferase GALNT2 2 Q10472 Polypeptide N-acetylgalactosaminyltransferase GALNT1 1 Q11201 CMP-N-acetylneuraminate-beta- ST3GAL1 galactosamide-alpha-2,3-sialyltransferase 1 Q11203 CMP-N-acetylneuraminate-beta-1,4- ST3GAL3 galactoside alpha-2,3-sialyltransferase Q11206 CMP-N-acetylneuraminate-beta- ST3GAL4 galactosamide-alpha-2,3-sialyltransferase 4 Q12794 Hyaluronidase-1 HYAL1 Q12805 EGF-containing fibulin-like extracellular matrix EFEMP1 protein 1 Q12836 Zona pellucida sperm-binding protein 4 ZP4 Q12841 Follistatin-related protein 1 FSTL1 Q12904 Aminoacyl tRNA synthase complex-interacting AIMP1 multifunctional protein 1 Q13018 Soluble secretory phospholipase A2 receptor PLA2R1 Q13072 B melanoma antigen 1 BAGE Q13093 Platelet-activating factor acetylhydrolase PLA2G7 Q13103 Secreted phosphoprotein 24 SPP2 Q13162 Peroxiredoxin-4 PRDX4 Q13201 Platelet glycoprotein Ia* MMRN1 Q13214 Semaphorin-3B SEMA3B Q13219 Pappalysin-1 PAPPA Q13231 Chitotriosidase-1 CHIT1 Q13253 Noggin NOG Q13261 Interleukin-15 receptor subunit alpha IL15RA Q13275 Semaphorin-3F SEMA3F Q13291 Signaling lymphocytic activation molecule SLAMF1 Q13316 Dentin matrix acidic phosphoprotein 1 DMP1 Q13361 Microfibrillar-associated protein 5 MFAP5 Q13410 Butyrophilin subfamily 1 member A1 BTN1A1 Q13421 Mesothelin, cleaved form MSLN Q13429 Insulin-like growth factor I IGF-I Q13443 Disintegrin and metalloproteinase domain- ADAM9 containing protein 9 Q13519 Neuropeptide 1 PNOC Q13751 Laminin subunit beta-3 LAMB3 Q13753 Laminin subunit gamma-2 LAMC2 Q13790 Apolipoprotein F APOF Q13822 Ectonucleotide pyrophosphatase/ ENPP2 phosphodiesterase family member 2 Q14031 Collagen alpha-6(IV) chain COL4A6 Q14050 Collagen alpha-3(IX) chain COL9A3 Q14055 Collagen alpha-2(IX) chain COL9A2 Q14112 Nidogen-2 NID2 Q14114 Low-density lipoprotein receptor-related LRP8 protein 8 Q14118 Dystroglycan DAG1 Q14314 Fibroleukin FGL2 Q14393 Growth arrest-specific protein 6 GAS6 Q14406 Chorionic somatomammotropin hormone-like CSHL1 1 Q14507 Epididymal secretory protein E3-alpha EDDM3A Q14508 WAP four-disulfide core domain protein 2 WFDC2 Q14512 Fibroblast growth factor-binding protein 1 FGFBP1 Q14515 SPARC-like protein 1 SPARCL1 Q14520 Hyaluronan-binding protein 2 27 kDa light HABP2 chain Q14563 Semaphorin-3A SEMA3A Q14623 Indian hedgehog protein IHH Q14624 Inter-alpha-trypsin inhibitor heavy chain H4 ITIH4 Q14667 UPF0378 protein KIAA0100 KIAA0100 Q14703 Membrane-bound transcription factor site-1 MBTPS1 protease Q14766 Latent-transforming growth factor beta- LTBP1 binding protein 1 Q14767 Latent-transforming growth factor beta- LTBP2 binding protein 2 Q14773 Intercellular adhesion molecule 4 ICAM4 Q14993 Collagen alpha-1(XIX) chain COL19A1 Q14CN2 Calcium-activated chloride channel regulator 4, CLCA4 110 kDa form Q15046 Lysine--tRNA ligase KARS Q15063 Periostin POSTN Q15109 Advanced glycosylation end product-specific AGER receptor Q15113 Procollagen C-endopeptidase enhancer 1 PCOLCE Q15166 Serum paraoxonase/lactonase 3 PON3 Q15195 Plasminogen-like protein A PLGLA Q15198 Platelet-derived growth factor receptor-like PDGFRL protein Q15223 Poliovirus receptor-related protein 1 PVRL1 Q15238 Pregnancy-specific beta-1-glycoprotein 5 PSG5 Q15363 Transmembrane emp24 domain-containing TMED2 protein 2 Q15375 Ephrin type-A receptor 7 EPHA7 Q15389 Angiopoietin-1 ANGPT1 Q15465 Sonic hedgehog protein SHH Q15485 Ficolin-2 FCN2 Q15517 Corneodesmosin CDSN Q15582 Transforming growth factor-beta-induced TGFBI protein ig-h3 Q15661 Tryptase alpha/beta-1 TPSAB1 Q15726 Metastin KISS1 Q15782 Chitinase-3-like protein 2 CHI3L2 Q15828 Cystatin-M CST6 Q15846 Clusterin-like protein 1 CLUL1 Q15848 Adiponectin ADIPOQ Q16206 Protein disulfide-thiol oxidoreductase ENOX2 Q16270 Insulin-like growth factor-binding protein 7 IGFBP7 Q16363 Laminin subunit alpha-4 LAMA4 Q16378 Proline-rich protein 4 PRR4 Q16557 Pregnancy-specific beta-1-glycoprotein 3 PSG3 Q16568 CART(42-89) CARTPT Q16610 Extracellular matrix protein 1 ECM1 Q16619 Cardiotrophin-1 CTF1 Q16623 Syntaxin-1A STX1A Q16627 HCC-1(9-74) CCL14 Q16651 Prostasin light chain PRSS8 Q16661 Guanylate cyclase C-activating peptide 2 GUCA2B Q16663 CCL15(29-92) CCL15 Q16674 Melanoma-derived growth regulatory protein MIA Q16769 Glutaminyl-peptide cyclotransferase QPCT Q16787 Laminin subunit alpha-3 LAMA3 Q16842 CMP-N-acetylneuraminate-beta- ST3GAL2 galactosamide-alpha-2,3-sialyltransferase 2 Q17RR3 Pancreatic lipase-related protein 3 PNLIPRP3 Q17RW2 Collagen alpha-1(XXIV) chain COL24A1 Q17RY6 Lymphocyte antigen 6K LY6K Q1L6U9 Prostate-associated microseminoprotein MSMP Q1W4C9 Serine protease inhibitor Kazal-type 13 SPINK13 Q1ZYL8 Izumo sperm-egg fusion protein 4 IZUMO4 Q29960 HLA class I histocompatibility antigen, Cw-16 HLA-C alpha chain Q2I0M5 R-spondin-4 RSPO4 Q2L4Q9 Serine protease 53 PRSS53 Q2MKA7 R-spondin-1 RSPO1 Q2MV58 Tectonic-1 TCTN1 Q2TAL6 Brorin VWC2 Q2UY09 Collagen alpha-1(XXVIII) chain COL28A1 Q2VPA4 Complement component receptor 1-like CR1L protein Q2WEN9 Carcinoembryonic antigen-related cell CEACAM16 adhesion molecule 16 Q30KP8 Beta-defensin 136 DEFB136 Q30KP9 Beta-defensin 135 DEFB135 Q30KQ1 Beta-defensin 133 DEFB133 Q30KQ2 Beta-defensin 130 DEFB130 Q30KQ4 Beta-defensin 116 DEFB116 Q30KQ5 Beta-defensin 115 DEFB115 Q30KQ6 Beta-defensin 114 DEFB114 Q30KQ7 Beta-defensin 113 DEFB113 Q30KQ8 Beta-defensin 112 DEFB112 Q30KQ9 Beta-defensin 110 DEFB110 Q30KR1 Beta-defensin 109 DEFB109P1 Q32P28 Prolyl 3-hydroxylase 1 LEPRE1 Q3B7J2 Glucose-fructose oxidoreductase domain- GFOD2 containing protein 2 Q3SY79 Protein Wnt WNT3A Q3T906 N-acetylglucosamine-1-phosphotransferase GNPTAB subunits alpha/beta Q495T6 Membrane metallo-endopeptidase-like 1 MMEL1 Q49AH0 Cerebral dopamine neurotrophic factor CDNF Q4G0G5 Secretoglobin family 2B member 2 SCGB2B2 Q4G0M1 Protein FAM132B FAM132B Q4LDE5 Sushi, von Willebrand factor type A, EGF and SVEP1 pentraxin domain-containing protein 1 Q4QY38 Beta-defensin 134 DEFB134 Q4VAJ4 Protein Wnt WNT10B Q4W5P6 Protein TMEM155 TMEM155 Q4ZHG4 Fibronectin type III domain-containing protein FNDC1 1 Q53H76 Phospholipase A1 member A PLA1A Q53RD9 Fibulin-7 FBLN7 Q53S33 BolA-like protein 3 BOLA3 Q5BLP8 Neuropeptide-like protein C4orf48 C4orf48 Q5DT21 Serine protease inhibitor Kazal-type 9 SPINK9 Q5EBL8 PDZ domain-containing protein 11 PDZD11 Q5FYB0 Arylsulfatase J ARSJ Q5FYB1 Arylsulfatase I ARSI Q5GAN3 Ribonuclease-like protein 13 RNASE13 Q5GAN4 Ribonuclease-like protein 12 RNASE12 Q5GAN6 Ribonuclease-like protein 10 RNASE10 Q5GFL6 von Willebrand factor A domain-containing VWA2 protein 2 Q5H8A3 Neuromedin-S NMS Q5H8C1 FRAS1-related extracellular matrix protein 1 FREM1 Q5IJ48 Protein crumbs homolog 2 CRB2 Q5J5C9 Beta-defensin 121 DEFB121 Q5JS37 NHL repeat-containing protein 3 NHLRC3 Q5JTB6 Placenta-specific protein 9 PLAC9 Q5JU69 Torsin-2A TOR2A Q5JXM2 Methyltransferase-like protein 24 METTL24 Q5JZY3 Ephrin type-A receptor 10 EPHA10 Q5K4E3 Polyserase-2 PRSS36 Q5SRR4 Lymphocyte antigen 6 complex locus protein LY6G5C G5c Q5T1H1 Protein eyes shut homolog EYS Q5T4F7 Secreted frizzled-related protein 5 SFRP5 Q5T4W7 Artemin ARTN Q5T7M4 Protein FAM132A FAM132A Q5TEH8 Protein Wnt WNT2B Q5TIE3 von Willebrand factor A domain-containing VWA5B1 protein 5B1 Q5UCC4 ER membrane protein complex subunit 10 EMC10 Q5VST6 Abhydrolase domain-containing protein FAM108B1 FAM108B1 Q5VTL7 Fibronectin type III domain-containing protein FNDC7 7 Q5VUM1 UPF0369 protein C6orf57 C6orf57 Q5VV43 Dyslexia-associated protein KIAA0319 KIAA0319 Q5VWW1 Complement C1q-like protein 3 C1QL3 Q5VXI9 Lipase member N LIPN Q5VXJ0 Lipase member K LIPK Q5VXM1 CUB domain-containing protein 2 CDCP2 Q5VYX0 Renalase RNLS Q5VYY2 Lipase member M LIPM Q5W186 Cystatin-9 CST9 Q5W5W9 Regulated endocrine-specific protein 18 RESP18 Q5XG92 Carboxylesterase 4A CES4A Q63HQ2 Pikachurin EGFLAM Q641Q3 Meteorin-like protein METRNL Q66K79 Carboxypeptidase Z CPZ Q685J3 Mucin-17 MUC17 Q68BL7 Olfactomedin-like protein 2A OLFML2A Q68BL8 Olfactomedin-like protein 2B OLFML2B Q68DV7 E3 ubiquitin-protein ligase RNF43 RNF43 Q6B9Z1 Insulin growth factor-like family member 4 IGFL4 Q6BAA4 Fc receptor-like B FCRLB Q6E0U4 Dermokine DMKN Q6EMK4 Vasorin VASN Q6FHJ7 Secreted frizzled-related protein 4 SFRP4 Q6GPI1 Chymotrypsin B2 chain B CTRB2 Q6GTS8 Probable carboxypeptidase PM20D1 PM20D1 Q6H9L7 Isthmin-2 ISM2 Q6IE36 Ovostatin homolog 2 OVOS2 Q6IE37 Ovostatin homolog 1 OVOS1 Q6IE38 Serine protease inhibitor Kazal-type 14 SPINK14 Q6ISS4 Leukocyte-associated immunoglobulin-like LAIR2 receptor 2 Q6JVE5 Epididymal-specific lipocalin-12 LCN12 Q6JVE6 Epididymal-specific lipocalin-10 LCN10 Q6JVE9 Epididymal-specific lipocalin-8 LCN8 Q6KF10 Growth/differentiation factor 6 GDF6 Q6MZW2 Follistatin-related protein 4 FSTL4 Q6NSX1 Coiled-coil domain-containing protein 70 CCDC70 Q6NT32 Carboxylesterase 5A CES5A Q6NT52 Choriogonadotropin subunit beta variant 2 CGB2 Q6NUI6 Chondroadherin-like protein CHADL Q6NUJ1 Saposin A-like PSAPL1 Q6P093 Arylacetamide deacetylase-like 2 AADACL2 Q6P4A8 Phospholipase B-like 1 PLBD1 Q6P5S2 UPF0762 protein C6orf58 C6orf58 Q6P988 Protein notum homolog NOTUM Q6PCB0 von Willebrand factor A domain-containing VWA1 protein 1 Q6PDA7 Sperm-associated antigen 11A SPAG11A Q6PEW0 Inactive serine protease 54 PRSS54 Q6PEZ8 Podocan-like protein 1 PODNL1 Q6PKH6 Dehydrogenase/reductase SDR family member DHRS4L2 4-like 2 Q6Q788 Apolipoprotein A-V APOA5 Q6SPF0 Atherin SAMD1 Q6UDR6 Kunitz-type protease inhibitor 4 SPINT4 Q6URK8 Testis, prostate and placenta-expressed protein TEPP Q6UW01 Cerebellin-3 CBLN3 Q6UW10 Surfactant-associated protein 2 SFTA2 Q6UW15 Regenerating islet-derived protein 3-gamma REG3G Q6UW32 Insulin growth factor-like family member 1 IGFL1 Q6UW78 UPF0723 protein C11orf83 C11orf83 Q6UW88 Epigen EPGN Q6UWE3 Colipase-like protein 2 CLPSL2 Q6UWF7 NXPE family member 4 NXPE4 Q6UWF9 Protein FAM180A FAM180A Q6UWM5 GLIPR1-like protein 1 GLIPR1L1 Q6UWN8 Serine protease inhibitor Kazal-type 6 SPINK6 Q6UWP2 Dehydrogenase/reductase SDR family member DHRS11 11 Q6UWP8 Suprabasin SBSN Q6UWQ5 Lysozyme-like protein 1 LYZL1 Q6UWQ7 Insulin growth factor-like family member 2 IGFL2 Q6UWR7 Ectonucleotide pyrophosphatase/ ENPP6 phosphodiesterase family member 6 soluble form Q6UWT2 Adropin ENHO Q6UWU2 Beta-galactosidase-1-like protein GLB1L Q6UWW0 Lipocalin-15 LCN15 Q6UWX4 HHIP-like protein 2 HHIPL2 Q6UWY0 Arylsulfatase K ARSK Q6UWY2 Serine protease 57 PRSS57 Q6UWY5 Olfactomedin-like protein 1 OLFML1 Q6UX06 Olfactomedin-4 OLFM4 Q6UX07 Dehydrogenase/reductase SDR family member DHRS13 13 Q6UX39 Amelotin AMTN Q6UX46 Protein FAM150B FAM150B Q6UX73 UPF0764 protein C16orf89 C16orf89 Q6UXB0 Protein FAM131A FAM131A Q6UXB1 Insulin growth factor-like family member 3 IGFL3 Q6UXB2 VEGF co-regulated chemokine 1 CXCL17 Q6UXF7 C-type lectin domain family 18 member B CLEC18B Q6UXH0 Hepatocellular carcinoma-associated protein C19orf80 TD26 Q6UXH1 Cysteine-rich with EGF-like domain protein 2 CRELD2 Q6UXH8 Collagen and calcium-binding EGF domain- CCBE1 containing protein 1 Q6UXH9 Inactive serine protease PAMR1 PAMR1 Q6UXI7 Vitrin VIT Q6UXI9 Nephronectin NPNT Q6UXN2 Trem-like transcript 4 protein TREML4 Q6UXS0 C-type lectin domain family 19 member A CLEC19A Q6UXT8 Protein FAM150A FAM150A Q6UXT9 Abhydrolase domain-containing protein 15 ABHD15 Q6UXV4 Apolipoprotein O-like APOOL Q6UXX5 Inter-alpha-trypsin inhibitor heavy chain H6 ITIH6 Q6UXX9 R-spondin-2 RSPO2 Q6UY14 ADAMTS-like protein 4 ADAMTSL4 Q6UY27 Prostate and testis expressed protein 2 PATE2 Q6W4X9 Mucin-6 MUC6 Q6WN34 Chordin-like protein 2 CHRDL2 Q6WRI0 Immunoglobulin superfamily member 10 IGSF10 Q6X4U4 Sclerostin domain-containing protein 1 SOSTDC1 Q6X784 Zona pellucida-binding protein 2 ZPBP2 Q6XE38 Secretoglobin family 1D member 4 SCGB1D4 Q6XPR3 Repetin RPTN Q6XZB0 Lipase member I LIPI Q6ZMM2 ADAMTS-like protein 5 ADAMTSL5 Q6ZMP0 Thrombospondin type-1 domain-containing THSD4 protein 4 Q6ZNF0 Iron/zinc purple acid phosphatase-like protein PAPL Q6ZRI0 Otogelin OTOG Q6ZRP7 Sulfhydryl oxidase 2 QSOX2 Q6ZWJ8 Kielin/chordin-like protein KCP Q75N90 Fibrillin-3 FBN3 Q765I0 Urotensin-2B UTS2D Q76B58 Protein FAM5C FAM5C Q76LX8 A disintegrin and metalloproteinase with ADAMTS13 thrombospondin motifs 13 Q76M96 Coiled-coil domain-containing protein 80 CCDC80 Q7L1S5 Carbohydrate sulfotransferase 9 CHST9 Q7L513 Fc receptor-like A FCRLA Q7L8A9 Vasohibin-1 VASH1 Q7RTM1 Otopetrin-1 OTOP1 Q7RTW8 Otoancorin OTOA Q7RTY5 Serine protease 48 PRSS48 Q7RTY7 Ovochymase-1 OVCH1 Q7RTZ1 Ovochymase-2 OVCH2 Q7Z304 MAM domain-containing protein 2 MAMDC2 Q7Z3S9 Notch homolog 2 N-terminal-like protein NOTCH2NL Q7Z4H4 Intermedin-short ADM2 Q7Z4P5 Growth/differentiation factor 7 GDF7 Q7Z4R8 UPF0669 protein C6orf120 C6orf120 Q7Z4W2 Lysozyme-like protein 2 LYZL2 Q7Z5A4 Serine protease 42 PRSS42 Q7Z5A7 Protein FAM19A5 FAM19A5 Q7Z5A8 Protein FAM19A3 FAM19A3 Q7Z5A9 Protein FAM19A1 FAM19A1 Q7Z5J1 Hydroxysteroid 11-beta-dehydrogenase 1-like HSD11B1L protein Q7Z5L0 Vitelline membrane outer layer protein 1 VMO1 homolog Q7Z5L3 Complement C1q-like protein 2 C1QL2 Q7Z5L7 Podocan PODN Q7Z5P4 17-beta-hydroxysteroid dehydrogenase 13 HSD17B13 Q7Z5P9 Mucin-19 MUC19 Q7Z5Y6 Bone morphogenetic protein 8A BMP8A Q7Z7B7 Beta-defensin 132 DEFB132 Q7Z7B8 Beta-defensin 128 DEFB128 Q7Z7C8 Transcription initiation factor TFIID subunit 8 TAF8 Q7Z7H5 Transmembrane emp24 domain-containing TMED4 protein 4 Q86SG7 Lysozyme g-like protein 2 LYG2 Q86SI9 Protein CEI C5orf38 Q86TE4 Leucine zipper protein 2 LUZP2 Q86TH1 ADAMTS-like protein 2 ADAMTSL2 Q86U17 Serpin A11 SERPINA11 Q86UU9 Endokinin-A TAC4 Q86UW8 Hyaluronan and proteoglycan link protein 4 HAPLN4 Q86UX2 Inter-alpha-trypsin inhibitor heavy chain H5 ITIH5 Q86V24 Adiponectin receptor protein 2 ADIPOR2 Q86VB7 Soluble CD163 CD163 Q86VR8 Four-jointed box protein 1 FJX1 Q86WD7 Serpin A9 SERPINA9 Q86WN2 Interferon epsilon IFNE Q86WS3 Placenta-specific 1-like protein PLAC1L Q86X52 Chondroitin sulfate synthase 1 CHSY1 Q86XP6 Gastrokine-2 GKN2 Q86XS5 Angiopoietin-related protein 5 ANGPTL5 Q86Y27 B melanoma antigen 5 BAGE5 Q86Y28 B melanoma antigen 4 BAGE4 Q86Y29 B melanoma antigen 3 BAGE3 Q86Y30 B melanoma antigen 2 BAGE2 Q86Y38 Xylosyltransferase 1 XYLT1 Q86Y78 Ly6/PLAUR domain-containing protein 6 LYPD6 Q86YD3 Transmembrane protein 25 TMEM25 Q86YJ6 Threonine synthase-like 2 THNSL2 Q86YW7 Glycoprotein hormone beta-5 GPHB5 Q86Z23 Complement C1q-like protein 4 C1QL4 Q8IU57 Interleukin-28 receptor subunit alpha IL28RA Q8IUA0 WAP four-disulfide core domain protein 8 WFDC8 Q8IUB2 WAP four-disulfide core domain protein 3 WFDC3 Q8IUB3 Protein WFDC10B WFDC10B Q8IUB5 WAP four-disulfide core domain protein 13 WFDC13 Q8IUH2 Protein CREG2 CREG2 Q8IUK5 Plexin domain-containing protein 1 PLXDC1 Q8IUL8 Cartilage intermediate layer protein 2 C2 CILP2 Q8IUX7 Adipocyte enhancer-binding protein 1 AEBP1 Q8IUX8 Epidermal growth factor-like protein 6 EGFL6 Q8IVL8 Carboxypeptidase O CPO Q8IVN8 Somatomedin-B and thrombospondin type-1 SBSPON domain-containing protein Q8IVW8 Protein spinster homolog 2 SPNS2 Q8IW75 Serpin A12 SERPINA12 Q8IW92 Beta-galactosidase-1-like protein 2 GLB1L2 Q8IWL1 Pulmonary surfactant-associated protein A2 SFTPA2 Q8IWL2 Pulmonary surfactant-associated protein A1 SFTPA1 Q8IWV2 Contactin-4 CNTN4 Q8IWY4 Signal peptide, CUB and EGF-like domain- SCUBE1 containing protein 1 Q8IX30 Signal peptide, CUB and EGF-like domain- SCUBE3 containing protein 3 Q8IXA5 Sperm acrosome membrane-associated protein SPACA3 3, membrane form Q8IXB1 DnaJ homolog subfamily C member 10 DNAJC10 Q8IXL6 Extracellular serine/threonine protein kinase FAM20C Fam20C Q8IYD9 Lung adenoma susceptibility protein 2 LAS2 Q8IYP2 Serine protease 58 PRSS58 Q8IYS5 Osteoclast-associated immunoglobulin-like OSCAR receptor Q8IZC6 Collagen alpha-1(XXVII) chain COL27A1 Q8IZJ3 C3 and PZP-like alpha-2-macroglobulin domain- CPAMD8 containing protein 8 Q8IZN7 Beta-defensin 107 DEFB107B Q8N0V4 Leucine-rich repeat LGI family member 2 LGI2 Q8N104 Beta-defensin 106 DEFB106B Q8N119 Matrix metalloproteinase-21 MMP21 Q8N129 Protein canopy homolog 4 CNPY4 Q8N135 Leucine-rich repeat LGI family member 4 LGI4 Q8N145 Leucine-rich repeat LGI family member 3 LGI3 Q8N158 Glypican-2 GPC2 Q8N1E2 Lysozyme g-like protein 1 LYG1 Q8N2E2 von Willebrand factor D and EGF domain- VWDE containing protein Q8N2E6 Prosalusin TOR2A Q8N2S1 Latent-transforming growth factor beta- LTBP4 binding protein 4 Q8N302 Angiogenic factor with G patch and FHA AGGF1 domains 1 Q8N307 Mucin-20 MUC20 Q8N323 NXPE family member 1 NXPE1 Q8N387 Mucin-15 MUC15 Q8N3Z0 Inactive serine protease 35 PRSS35 Q8N436 Inactive carboxypeptidase-like protein X2 CPXM2 Q8N474 Secreted frizzled-related protein 1 SFRP1 Q8N475 Follistatin-related protein 5 FSTL5 Q8N4F0 BPI fold-containing family B member 2 BPIFB2 Q8N4T0 Carboxypeptidase A6 CPA6 Q8N5W8 Protein FAM24B FAM24B Q8N687 Beta-defensin 125 DEFB125 Q8N688 Beta-defensin 123 DEFB123 Q8N690 Beta-defensin 119 DEFB119 Q8N6C5 Immunoglobulin superfamily member 1 IGSF1 Q8N6C8 Leukocyte immunoglobulin-like receptor LILRA3 subfamily A member 3 Q8N6G6 ADAMTS-like protein 1 ADAMTSL1 Q8N6Y2 Leucine-rich repeat-containing protein 17 LRRC17 Q8N729 Neuropeptide W-23 NPW Q8N8U9 BMP-binding endothelial regulator protein BMPER Q8N907 DAN domain family member 5 DAND5 Q8NAT1 Glycosyltransferase-like domain-containing GTDC2 protein 2 Q8NAU1 Fibronectin type III domain-containing protein FNDC5 5 Q8NB37 Parkinson disease 7 domain-containing protein PDDC1 1 Q8NBI3 Draxin DRAXIN Q8NBM8 Prenylcysteine oxidase-like PCYOX1L Q8NBP7 Proprotein convertase subtilisin/kexin type 9 PCSK9 Q8NBQ5 Estradiol 17-beta-dehydrogenase 11 HSD17B11 Q8NBV8 Synaptotagmin-8 SYT8 Q8NCC3 Group XV phospholipase A2 PLA2G15 Q8NCF0 C-type lectin domain family 18 member C CLEC18C Q8NCW5 NAD(P)H-hydrate epimerase APOA1BP Q8NDA2 Hemicentin-2 HMCN2 Q8NDX9 Lymphocyte antigen 6 complex locus protein LY6G5B G5b Q8NDZ4 Deleted in autism protein 1 C3orf58 Q8NEB7 Acrosin-binding protein ACRBP Q8NES8 Beta-defensin 124 DEFB124 Q8NET1 Beta-defensin 108B DEFB108B Q8NEX5 Protein WFDC9 WFDC9 Q8NEX6 Protein WFDC11 WFDC11 Q8NF86 Serine protease 33 PRSS33 Q8NFM7 Interleukin-17 receptor D IL17RD Q8NFQ5 BPI fold-containing family B member 6 BPIFB6 Q8NFQ6 BPI fold-containing family C protein BPIFC Q8NFU4 Follicular dendritic cell secreted peptide FDCSP Q8NFW1 Collagen alpha-1(XXII) chain COL22A1 Q8NG35 Beta-defensin 105 DEFB105B Q8NG41 Neuropeptide B-23 NPB Q8NHW6 Otospiralin OTOS Q8NI99 Angiopoietin-related protein 6 ANGPTL6 Q8TAA1 Probable ribonuclease 11 RNASE11 Q8TAG5 V-set and transmembrane domain-containing VSTM2A protein 2A Q8TAL6 Fin bud initiation factor homolog FIBIN Q8TAT2 Fibroblast growth factor-binding protein 3 FGFBP3 Q8TAX7 Mucin-7 MUC7 Q8TB22 Spermatogenesis-associated protein 20 SPATA20 Q8TB73 Protein NDNF NDNF Q8TB96 T-cell immunomodulatory protein ITFG1 Q8TC92 Protein disulfide-thiol oxidoreductase ENOX1 Q8TCV5 WAP four-disulfide core domain protein 5 WFDC5 Q8TD06 Anterior gradient protein 3 homolog AGR3 Q8TD33 Secretoglobin family 1C member 1 SCGB1C1 Q8TD46 Cell surface glycoprotein CD200 receptor 1 CD200R1 Q8TDE3 Ribonuclease 8 RNASE8 Q8TDF5 Neuropilin and tolloid-like protein 1 NETO1 Q8TDL5 BPI fold-containing family B member 1 BPIFB1 Q8TE56 A disintegrin and metalloproteinase with ADAMTS17 thrombospondin motifs 17 Q8TE57 A disintegrin and metalloproteinase with ADAMTS16 thrombospondin motifs 16 Q8TE58 A disintegrin and metalloproteinase with ADAMTS15 thrombospondin motifs 15 Q8TE59 A disintegrin and metalloproteinase with ADAMTS19 thrombospondin motifs 19 Q8TE60 A disintegrin and metalloproteinase with ADAMTS18 thrombospondin motifs 18 Q8TE99 Acid phosphatase-like protein 2 ACPL2 Q8TER0 Sushi, nidogen and EGF-like domain-containing SNED1 protein 1 Q8TEU8 WAP, kazal, immunoglobulin, kunitz and NTR WFIKKN2 domain-containing protein 2 Q8WTQ1 Beta-defensin 104 DEFB104B Q8WTR8 Netrin-5 NTN5 Q8WTU2 Scavenger receptor cysteine-rich domain- SRCRB4D containing group B protein Q8WU66 Protein TSPEAR TSPEAR Q8WUA8 Tsukushin TSKU Q8WUF8 Protein FAM172A FAM172A Q8WUJ1 Neuferricin CYB5D2 Q8WUY1 UPF0670 protein THEM6 THEM6 Q8WVN6 Secreted and transmembrane protein 1 SECTM1 Q8WVQ1 Soluble calcium-activated nucleotidase 1 CANT1 Q8WWA0 Intelectin-1 ITLN1 Q8WWG1 Neuregulin-4 NRG4 Q8WWQ2 Inactive heparanase-2 HPSE2 Q8WWU7 Intelectin-2 ITLN2 Q8WWY7 WAP four-disulfide core domain protein 12 WFDC12 Q8WWY8 Lipase member H LIPH Q8WWZ8 Oncoprotein-induced transcript 3 protein OIT3 Q8WX39 Epididymal-specific lipocalin-9 LCN9 Q8WXA2 Prostate and testis expressed protein 1 PATE1 Q8WXD2 Secretogranin-3 SCG3 Q8WXF3 Relaxin-3 A chain RLN3 Q8WXI7 Mucin-16 MUC16 Q8WXQ8 Carboxypeptidase A5 CPA5 Q8WXS8 A disintegrin and metalloproteinase with ADAMTS14 thrombospondin motifs 14 Q92484 Acid sphingomyelinase-like phosphodiesterase SMPDL3A 3a Q92485 Acid sphingomyelinase-like phosphodiesterase SMPDL3B 3b Q92496 Complement factor H-related protein 4 CFHR4 Q92520 Protein FAM3C FAM3C Q92563 Testican-2 SPOCK2 Q92583 C-C motif chemokine 17 CCL17 Q92626 Peroxidasin homolog PXDN Q92743 Serine protease HTRA1 HTRA1 Q92752 Tenascin-R TNR Q92765 Secreted frizzled-related protein 3 FRZB Q92819 Hyaluronan synthase 2 HAS2 Q92820 Gamma-glutamyl hydrolase GGH Q92824 Proprotein convertase subtilisin/kexin type 5 PCSK5 Q92832 Protein kinase C-binding protein NELL1 NELL1 Q92838 Ectodysplasin-A, membrane form EDA Q92874 Deoxyribonuclease-1-like 2 DNASE1L2 Q92876 Kallikrein-6 KLK6 Q92913 Fibroblast growth factor 13 FGF13 Q92954 Proteoglycan 4 C-terminal part PRG4 Q93038 Tumor necrosis factor receptor superfamily TNFRSF25 member 25 Q93091 Ribonuclease K6 RNASE6 Q93097 Protein Wnt-2b WNT2B Q93098 Protein Wnt-8b WNT8B Q95460 Major histocompatibility complex class I- MR1 related gene protein Q969D9 Thymic stromal lymphopoietin TSLP Q969E1 Liver-expressed antimicrobial peptide 2 LEAP2 Q969H8 UPF0556 protein C19orf10 C19orf10 Q969Y0 NXPE family member 3 NXPE3 Q96A54 Adiponectin receptor protein 1 ADIPOR1 Q96A83 Collagen alpha-1(XXVI) chain EMID2 Q96A84 EMI domain-containing protein 1 EMID1 Q96A98 Tuberoinfundibular peptide of 39 residues PTH2 Q96A99 Pentraxin-4 PTX4 Q96BH3 Epididymal sperm-binding protein 1 ELSPBP1 Q96BQ1 Protein FAM3D FAM3D Q96CG8 Collagen triple helix repeat-containing protein CTHRC1 1 Q96DA0 Zymogen granule protein 16 homolog B ZG16B Q96DN2 von Willebrand factor C and EGF domain- VWCE containing protein Q96DR5 BPI fold-containing family A member 2 BPIFA2 Q96DR8 Mucin-like protein 1 MUCL1 Q96DX4 RING finger and SPRY domain-containing RSPRY1 protein 1 Q96EE4 Coiled-coil domain-containing protein 126 CCDC126 Q96GS6 Abhydrolase domain-containing protein FAM108A1 FAM108A1 Q96GW7 Brevican core protein BCAN Q96HF1 Secreted frizzled-related protein 2 SFRP2 Q96I82 Kazal-type serine protease inhibitor domain- KAZALD1 containing protein 1 Q96ID5 Immunoglobulin superfamily member 21 IGSF21 Q96II8 Leucine-rich repeat and calponin homology LRCH3 domain-containing protein 3 Q96IY4 Carboxypeptidase B2 CPB2 Q96JB6 Lysyl oxidase homolog 4 LOXL4 Q96JK4 HHIP-like protein 1 HHIPL1 Q96KN2 Beta-Ala-His dipeptidase CNDP1 Q96KW9 Protein SPACA7 SPACA7 Q96KX0 Lysozyme-like protein 4 LYZL4 Q96L15 Ecto-ADP-ribosyltransferase 5 ART5 Q96LB8 Peptidoglycan recognition protein 4 PGLYRP4 Q96LB9 Peptidoglycan recognition protein 3 PGLYRP3 Q96LC7 Sialic acid-binding Ig-like lectin 10 SIGLEC10 Q96LR4 Protein FAM19A4 FAM19A4 Q96MK3 Protein FAM20A FAM20A Q96MS3 Glycosyltransferase 1 domain-containing GLT1D1 protein 1 Q96NY8 Processed poliovirus receptor-related protein 4 PVRL4 Q96NZ8 WAP, kazal, immunoglobulin, kunitz and NTR WFIKKN1 domain-containing protein 1 Q96NZ9 Proline-rich acidic protein 1 PRAP1 Q96P44 Collagen alpha-1(XXI) chain COL21A1 Q96PB7 Noelin-3 OLFM3 Q96PC5 Melanoma inhibitory activity protein 2 MIA2 Q96PD5 N-acetylmuramoyl-L-alanine amidase PGLYRP2 Q96PH6 Beta-defensin 118 DEFB118 Q96PL1 Secretoglobin family 3A member 2 SCGB3A2 Q96PL2 Beta-tectorin TECTB Q96QH8 Sperm acrosome-associated protein 5 SPACA5 Q96QR1 Secretoglobin family 3A member 1 SCGB3A1 Q96QU1 Protocadherin-15 PCDH15 Q96QV1 Hedgehog-interacting protein HHIP Q96RW7 Hemicentin-1 HMCN1 Q96S42 Nodal homolog NODAL Q96S86 Hyaluronan and proteoglycan link protein 3 HAPLN3 Q96SL4 Glutathione peroxidase 7 GPX7 Q96SM3 Probable carboxypeptidase X1 CPXM1 Q96T91 Glycoprotein hormone alpha-2 GPHA2 Q99062 Granulocyte colony-stimulating factor receptor CSF3R Q99102 Mucin-4 alpha chain MUC4 Q99217 Amelogenin, X isoform AMELX Q99218 Amelogenin, Y isoform AMELY Q99435 Protein kinase C-binding protein NELL2 NELL2 Q99470 Stromal cell-derived factor 2 SDF2 Q99542 Matrix metalloproteinase-19 MMP19 Q99574 Neuroserpin SERPINI1 Q99584 Protein S100-A13 S100A13 Q99616 C-C motif chemokine 13 CCL13 Q99645 Epiphycan EPYC Q99674 Cell growth regulator with EF hand domain CGREF1 protein 1 Q99715 Collagen alpha-1(XII) chain COL12A1 Q99727 Metalloproteinase inhibitor 4 TIMP4 Q99731 C-C motif chemokine 19 CCL19 Q99748 Neurturin NRTN Q99935 Proline-rich protein 1 PROL1 Q99942 E3 ubiquitin-protein ligase RNF5 RNF5 Q99944 Epidermal growth factor-like protein 8 EGFL8 Q99954 Submaxillary gland androgen-regulated protein SMR3A 3A Q99969 Retinoic acid receptor responder protein 2 RARRES2 Q99972 Myocilin MYOC Q99983 Osteomodulin OMD Q99985 Semaphorin-3C SEMA3C Q99988 Growth/differentiation factor 15 GDF15 Q9BPW4 Apolipoprotein L4 APOL4 Q9BQ08 Resistin-like beta RETNLB Q9BQ16 Testican-3 SPOCK3 Q9BQ51 Programmed cell death 1 ligand 2 PDCD1LG2 Q9BQB4 Sclerostin SOST Q9BQI4 Coiled-coil domain-containing protein 3 CCDC3 Q9BQP9 BPI fold-containing family A member 3 BPIFA3 Q9BQR3 Serine protease 27 PRSS27 Q9BQY6 WAP four-disulfide core domain protein 6 WFDC6 Q9BRR6 ADP-dependent glucokinase ADPGK Q9BS86 Zona pellucida-binding protein 1 ZPBP Q9BSG0 Protease-associated domain-containing protein PRADC1 1 Q9BSG5 Retbindin RTBDN Q9BT30 Probable alpha-ketoglutarate-dependent ALKBH7 dioxygenase ABH7 Q9BT56 Spexin C12orf39 Q9BT67 NEDD4 family-interacting protein 1 NDFIP1 Q9BTY2 Plasma alpha-L-fucosidase FUCA2 Q9BU40 Chordin-like protein 1 CHRDL1 Q9BUD6 Spondin-2 SPON2 Q9BUN1 Protein MENT MENT Q9BUR5 Apolipoprotein O APOO Q9BV94 ER degradation-enhancing alpha-mannosidase- EDEM2 like 2 Q9BWP8 Collectin-11 COLEC11 Q9BWS9 Chitinase domain-containing protein 1 CHID1 Q9BX67 Junctional adhesion molecule C JAM3 Q9BX93 Group XIIB secretory phospholipase A2-like PLA2G12B protein Q9BXI9 Complement C1q tumor necrosis factor-related C1QTNF6 protein 6 Q9BXJ0 Complement C1q tumor necrosis factor-related C1QTNF5 protein 5 Q9BXJ1 Complement C1q tumor necrosis factor-related C1QTNF1 protein 1 Q9BXJ2 Complement C1q tumor necrosis factor-related C1QTNF7 protein 7 Q9BXJ3 Complement C1q tumor necrosis factor-related C1QTNF4 protein 4 Q9BXJ4 Complement C1q tumor necrosis factor-related C1QTNF3 protein 3 Q9BXJ5 Complement C1q tumor necrosis factor-related C1QTNF2 protein 2 Q9BXN1 Asporin ASPN Q9BXP8 Pappalysin-2 PAPPA2 Q9BXR6 Complement factor H-related protein 5 CFHR5 Q9BXS0 Collagen alpha-1(XXV) chain COL25A1 Q9BXX0 EMILIN-2 EMILIN2 Q9BXY4 R-spondin-3 RSPO3 Q9BY15 EGF-like module-containing mucin-like EMR3 hormone receptor-like 3 subunit beta Q9BY50 Signal peptidase complex catalytic subunit SEC11C SEC11C Q9BY76 Angiopoietin-related protein 4 ANGPTL4 Q9BYF1 Processed angiotensin-converting enzyme 2 ACE2 Q9BYJ0 Fibroblast growth factor-binding protein 2 FGFBP2 Q9BYW3 Beta-defensin 126 DEFB126 Q9BYX4 Interferon-induced helicase C domain- IFIH1 containing protein 1 Q9BYZ8 Regenerating islet-derived protein 4 REG4 Q9BZ76 Contactin-associated protein-like 3 CNTNAP3 Q9BZG9 Ly-6/neurotoxin-like protein 1 LYNX1 Q9BZJ3 Tryptase delta TPSD1 Q9BZM1 Group XIIA secretory phospholipase A2 PLA2G12A Q9BZM2 Group IIF secretory phospholipase A2 PLA2G2F Q9BZM5 NKG2D ligand 2 ULBP2 Q9BZP6 Acidic mammalian chitinase CHIA Q9BZZ2 Sialoadhesin SIGLEC1 Q9C0B6 Protein FAM5B FAM5B Q9GZM7 Tubulointerstitial nephritis antigen-like TINAGL1 Q9GZN4 Brain-specific serine protease 4 PRSS22 Q9GZP0 Platelet-derived growth factor D, receptor- PDGFD binding form Q9GZT5 Protein Wnt-10a WNT10A Q9GZU5 Nyctalopin NYX Q9GZV7 Hyaluronan and proteoglycan link protein 2 HAPLN2 Q9GZV9 Fibroblast growth factor 23 FGF23 Q9GZX9 Twisted gastrulation protein homolog 1 TWSG1 Q9GZZ7 GDNF family receptor alpha-4 GFRA4 Q9GZZ8 Extracellular glycoprotein lacritin LACRT Q9H0B8 Cysteine-rich secretory protein LCCL domain- CRISPLD2 containing 2 Q9H106 Signal-regulatory protein delta SIRPD Q9H114 Cystatin-like 1 CSTL1 Q9H173 Nucleotide exchange factor SIL1 SIL1 Q9H1E1 Ribonuclease 7 RNASE7 Q9H1F0 WAP four-disulfide core domain protein 10A WFDC10A Q9H1J5 Protein Wnt-8a WNT8A Q9H1J7 Protein Wnt-5b WNT5B Q9H1M3 Beta-defensin 129 DEFB129 Q9H1M4 Beta-defensin 127 DEFB127 Q9H1Z8 Augurin C2orf40 Q9H239 Matrix metalloproteinase-28 MMP28 Q9H2A7 C-X-C motif chemokine 16 CXCL16 Q9H2A9 Carbohydrate sulfotransferase 8 CHST8 Q9H2R5 Kallikrein-15 KLK15 Q9H2X0 Chordin CHRD Q9H2X3 C-type lectin domain family 4 member M CLEC4M Q9H306 Matrix metalloproteinase-27 MMP27 Q9H324 A disintegrin and metalloproteinase with ADAMTS10 thrombospondin motifs 10 Q9H336 Cysteine-rich secretory protein LCCL domain- CRISPLD1 containing 1 Q9H3E2 Sorting nexin-25 SNX25 Q9H3R2 Mucin-13 MUC13 Q9H3U7 SPARC-related modular calcium-binding SMOC2 protein 2 Q9H3Y0 Peptidase inhibitor R3HDML R3HDML Q9H4A4 Aminopeptidase B RNPEP Q9H4F8 SPARC-related modular calcium-binding SMOC1 protein 1 Q9H4G1 Cystatin-9-like CST9L Q9H5V8 CUB domain-containing protein 1 CDCP1 Q9H6B9 Epoxide hydrolase 3 EPHX3 Q9H6E4 Coiled-coil domain-containing protein 134 CCDC134 Q9H741 UPF0454 protein C12orf49 C12orf49 Q9H772 Gremlin-2 GREM2 Q9H7Y0 Deleted in autism-related protein 1 CXorf36 Q9H8L6 Multimerin-2 MMRN2 Q9H9S5 Fukutin-related protein FKRP Q9HAT2 Sialate O-acetylesterase SIAE Q9HB40 Retinoid-inducible serine carboxypeptidase SCPEP1 Q9HB63 Netrin-4 NTN4 Q9HBJ0 Placenta-specific protein 1 PLAC1 Q9HC23 Prokineticin-2 PROK2 Q9HC57 WAP four-disulfide core domain protein 1 WFDC1 Q9HC73 Cytokine receptor-like factor 2 CRLF2 Q9HC84 Mucin-5B MUC5B Q9HCB6 Spondin-1 SPON1 Q9HCQ7 Neuropeptide NPSF NPVF Q9HCT0 Fibroblast growth factor 22 FGF22 Q9HD89 Resistin RETN Q9NNX1 Tuftelin TUFT1 Q9NNX6 CD209 antigen CD209 Q9NP55 BPI fold-containing family A member 1 BPIFA1 Q9NP70 Ameloblastin AMBN Q9NP95 Fibroblast growth factor 20 FGF20 Q9NP99 Triggering receptor expressed on myeloid cells TREM1 1 Q9NPA2 Matrix metalloproteinase-25 MMP25 Q9NPE2 Neugrin NGRN Q9NPH0 Lysophosphatidic acid phosphatase type 6 ACP6 Q9NPH6 Odorant-binding protein 2b OBP2B Q9NQ30 Endothelial cell-specific molecule 1 ESM1 Q9NQ36 Signal peptide, CUB and EGF-like domain- SCUBE2 containing protein 2 Q9NQ38 Serine protease inhibitor Kazal-type 5 SPINK5 Q9NQ76 Matrix extracellular phosphoglycoprotein MEPE Q9NQ79 Cartilage acidic protein 1 CRTAC1 Q9NR16 Scavenger receptor cysteine-rich type 1 protein CD163L1 M160 Q9NR23 Growth/differentiation factor 3 GDF3 Q9NR71 Neutral ceramidase ASAH2 Q9NR99 Matrix-remodeling-associated protein 5 MXRA5 Q9NRA1 Platelet-derived growth factor C PDGFC Q9NRC9 Otoraplin OTOR Q9NRE1 Matrix metalloproteinase-26 MMP26 Q9NRJ3 C-C motif chemokine 28 CCL28 Q9NRM1 Enamelin ENAM Q9NRN5 Olfactomedin-like protein 3 OLFML3 Q9NRR1 Cytokine-like protein 1 CYTL1 Q9NS15 Latent-transforming growth factor beta- LTBP3 binding protein 3 Q9NS62 Thrombospondin type-1 domain-containing THSD1 protein 1 Q9NS71 Gastrokine-1 GKN1 Q9NS98 Semaphorin-3G SEMA3G Q9NSA1 Fibroblast growth factor 21 FGF21 Q9NT22 EMILIN-3 EMILIN3 Q9NTU7 Cerebellin-4 CBLN4 Q9NVR0 Kelch-like protein 11 KLHL11 Q9NWH7 Spermatogenesis-associated protein 6 SPATA6 Q9NXC2 Glucose-fructose oxidoreductase domain- GFOD1 containing protein 1 Q9NY56 Odorant-binding protein 2a OBP2A Q9NY84 Vascular non-inflammatory molecule 3 VNN3 Q9NZ20 Group 3 secretory phospholipase A2 PLA2G3 Q9NZC2 Triggering receptor expressed on myeloid cells TREM2 2 Q9NZK5 Adenosine deaminase CECR1 CECR1 Q9NZK7 Group IIE secretory phospholipase A2 PLA2G2E Q9NZP8 Complement C1r subcomponent-like protein C1RL Q9NZV1 Cysteine-rich motor neuron 1 protein CRIM1 Q9NZW4 Dentin sialoprotein DSPP Q9P0G3 Kallikrein-14 KLK14 Q9P0W0 Interferon kappa IFNK Q9P218 Collagen alpha-1(XX) chain COL20A1 Q9P2C4 Transmembrane protein 181 TMEM181 Q9P2K2 Thioredoxin domain-containing protein 16 TXNDC16 Q9P2N4 A disintegrin and metalloproteinase with ADAMTS9 thrombospondin motifs 9 Q9UBC7 Galanin-like peptide GALP Q9UBD3 Cytokine SCM-1 beta XCL2 Q9UBD9 Cardiotrophin-like cytokine factor 1 CLCF1 Q9UBM4 Opticin OPTC Q9UBP4 Dickkopf-related protein 3 DKK3 Q9UBQ6 Exostosin-like 2 EXTL2 Q9UBR5 Chemokine-like factor CKLF Q9UBS5 Gamma-aminobutyric acid type B receptor GABBR1 subunit 1 Q9UBT3 Dickkopf-related protein 4 short form DKK4 Q9UBU2 Dickkopf-related protein 2 DKK2 Q9UBU3 Ghrelin-28 GHRL Q9UBV4 Protein Wnt-16 WNT16 Q9UBX5 Fibulin-5 FBLN5 Q9UBX7 Kallikrein-11 KLK11 Q9UEF7 Klotho KL Q9UFP1 Protein FAM198A FAM198A Q9UGM3 Deleted in malignant brain tumors 1 protein DMBT1 Q9UGM5 Fetuin-B FETUB Q9UGP8 Translocation protein SEC63 homolog SEC63 Q9UHF0 Neurokinin-B TAC3 Q9UHF1 Epidermal growth factor-like protein 7 EGFL7 Q9UHG2 ProSAAS PCSK1N Q9UHI8 A disintegrin and metalloproteinase with ADAMTS1 thrombospondin motifs 1 Q9UHL4 Dipeptidyl peptidase 2 DPP7 Q9UI42 Carboxypeptidase A4 CPA4 Q9UIG4 Psoriasis susceptibility 1 candidate gene 2 PSORS1C2 protein Q9UIK5 Tomoregulin-2 TMEFF2 Q9UIQ6 Leucyl-cystinyl aminopeptidase, pregnancy LNPEP serum form Q9UJA9 Ectonucleotide ENPP5 pyrophosphatase/phosphodiesterase family member 5 Q9UJH8 Meteorin METRN Q9UJJ9 N-acetylglucosamine-1-phosphotransferase GNPTG subunit gamma Q9UJW2 Tubulointerstitial nephritis antigen TINAG Q9UK05 Growth/differentiation factor 2 GDF2 Q9UK55 Protein Z-dependent protease inhibitor SERPINA10 Q9UK85 Dickkopf-like protein 1 DKKL1 Q9UKJ1 Paired immunoglobulin-like type 2 receptor PILRA alpha Q9UKP4 A disintegrin and metalloproteinase with ADAMTS7 thrombospondin motifs 7 Q9UKP5 A disintegrin and metalloproteinase with ADAMTS6 thrombospondin motifs 6 Q9UKQ2 Disintegrin and metalloproteinase domain- ADAM28 containing protein 28 Q9UKQ9 Kallikrein-9 KLK9 Q9UKR0 Kallikrein-12 KLK12 Q9UKR3 Kallikrein-13 KLK13 Q9UKU9 Angiopoietin-related protein 2 ANGPTL2 Q9UKZ9 Procollagen C-endopeptidase enhancer 2 PCOLCE2 Q9UL52 Transmembrane protease serine 11E non- TMPRSS11E catalytic chain Q9ULC0 Endomucin EMCN Q9ULI3 Protein HEG homolog 1 HEG1 Q9ULZ1 Apelin-13 APLN Q9ULZ9 Matrix metalloproteinase-17 MMP17 Q9UM21 Alpha-1,3-mannosyl-glycoprotein 4-beta-N- MGAT4A acetylglucosaminyltransferase A soluble form Q9UM22 Mammalian ependymin-related protein 1 EPDR1 Q9UM73 ALK tyrosine kinase receptor ALK Q9UMD9 97 kDa linear IgA disease antigen COL17A1 Q9UMX5 Neudesin NENF Q9UN73 Protocadherin alpha-6 PCDHA6 Q9UNA0 A disintegrin and metalloproteinase with ADAMTS5 thrombospondin motifs 5 Q9UNI1 Chymotrypsin-like elastase family member 1 CELA1 Q9UNK4 Group IID secretory phospholipase A2 PLA2G2D Q9UP79 A disintegrin and metalloproteinase with ADAMTS8 thrombospondin motifs 8 Q9UPZ6 Thrombospondin type-1 domain-containing THSD7A protein 7A Q9UQ72 Pregnancy-specific beta-1-glycoprotein 11 PSG11 Q9UQ74 Pregnancy-specific beta-1-glycoprotein 8 PSG8 Q9UQC9 Calcium-activated chloride channel regulator 2 CLCA2 Q9UQE7 Structural maintenance of chromosomes SMC3 protein 3 Q9UQP3 Tenascin-N TNN Q9Y223 UDP-N-acetylglucosamine 2-epimerase GNE Q9Y240 C-type lectin domain family 11 member A CLEC11A Q9Y251 Heparanase 8 kDa subunit HPSE Q9Y258 C-C motif chemokine 26 CCL26 Q9Y264 Angiopoietin-4 ANGPT4 Q9Y275 Tumor necrosis factor ligand superfamily TNFSF13B member 13b, membrane form Q9Y287 BRI2 intracellular domain ITM2B Q9Y2E5 Epididymis-specific alpha-mannosidase MAN2B2 Q9Y334 von Willebrand factor A domain-containing VWA7 protein 7 Q9Y337 Kallikrein-5 KLK5 Q9Y3B3 Transmembrane emp24 domain-containing TMED7 protein 7 Q9Y3E2 BolA-like protein 1 BOLA1 Q9Y426 C2 domain-containing protein 2 C2CD2 Q9Y4K0 Lysyl oxidase homolog 2 LOXL2 Q9Y4X3 C-C motif chemokine 27 CCL27 Q9Y5C1 Angiopoietin-related protein 3 ANGPTL3 Q9Y5I2 Protocadherin alpha-10 PCDHA10 Q9Y5I3 Protocadherin alpha-1 PCDHA1 Q9Y5K2 Kallikrein-4 KLK4 Q9Y5L2 Hypoxia-inducible lipid droplet-associated HILPDA protein Q9Y5Q5 Atrial natriuretic peptide-converting enzyme CORIN Q9Y5R2 Matrix metalloproteinase-24 MMP24 Q9Y5U5 Tumor necrosis factor receptor superfamily TNFRSF18 member 18 Q9Y5W5 Wnt inhibitory factor 1 WIF1 Q9Y5X9 Endothelial lipase LIPG Q9Y625 Secreted glypican-6 GPC6 Q9Y646 Carboxypeptidase Q CPQ Q9Y6C2 EMILIN-1 EMILIN1 Q9Y6F9 Protein Wnt-6 WNT6 Q9Y6I9 Testis-expressed sequence 264 protein TEX264 Q9Y6L7 Tolloid-like protein 2 TLL2 Q9Y6N3 Calcium-activated chloride channel regulator CLCA3P family member 3 Q9Y6N6 Laminin subunit gamma-3 LAMC3 Q9Y6R7 IgGFc-binding protein FCGBP Q9Y6Y9 Lymphocyte antigen 96 LY96 Q9Y6Z7 Collectin-10 COLEC10

In one set of embodiments, the MCNA compound comprises two encoding polynucleotides. For example, the MCNA compound may be a palindromic coding nucleic acid (PCNA) having two encoding polynucleotides each having a polynucleotide portion that codes for the same protein.

In some embodiments, a MCNA compound comprises an encoding polynucleotide that encodes Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR) mRNA, linked to a non-coding polynucleotide via a 3′ end linkage between the polynucleotides. In some embodiments, a MCNA compound comprises two or more encoding polynucleotides linked via a 3′ end linkage between the polynucleotides such that the MCNA compound comprises two or more 5′ ends, wherein at least one of the encoding polynucleotides encodes hCFTR. In some embodiments, a MCNA compound is a palindromic coding nucleic acid (PCNA) comprising two encoding polynucleotides linked via a 3′ end linkage between the polynucleotides such that the MCNA compound comprises two or more 5′ ends, wherein each encoding polynucleotide codes for hCFTR. In some embodiments, a MCNA compound comprises two or more polynucleotides linked via a 3′ end linkage between the polynucleotides such that the MCNA compound comprises two or more 5′ ends, wherein at least one polynucleotide is an encoding polynucleotide that encodes hCFTR and at least one polynucleotide acts as a protecting group.

In some embodiments, a MCNA compound comprises an encoding polynucleotide that encodes human phenylalanine hydroxylase (hPAH) mRNA, linked to a non-coding polynucleotide via a 3′ end linkage between the polynucleotides. In some embodiments, a MCNA compound comprises two or more encoding polynucleotides linked via a 3′ end linkage between the polynucleotides such that the MCNA compound comprises two or more 5′ ends, wherein at least one of the encoding polynucleotides encodes hPAH. In some embodiments, a MCNA compound is a palindromic coding nucleic acid (PCNA) comprising two encoding polynucleotides linked via a 3′ end linkage between the polynucleotides such that the MCNA compound comprises two or more 5′ ends, wherein each encoding polynucleotide codes for hPAH. In some embodiments, a MCNA compound comprises two or more polynucleotides linked via a 3′ end linkage between the polynucleotides such that the MCNA compound comprises two or more 5′ ends, wherein at least one polynucleotide is an encoding polynucleotide that encodes hPAH and at least one polynucleotide acts as a protecting group.

In some embodiments, a MCNA compound comprises an encoding polynucleotide that encodes human Ornithine transcarbamylase (hOTC) mRNA, linked to a non-coding polynucleotide via a 3′ end linkage between the polynucleotides. In some embodiments, a MCNA compound comprises two or more encoding polynucleotides linked via a 3′ end linkage between the polynucleotides such that the MCNA compound comprises two or more 5′ ends, wherein at least one of the encoding polynucleotides encodes hOTC. In some embodiments, a MCNA compound is a palindromic coding nucleic acid (PCNA) comprising two encoding polynucleotides linked via a 3′ end linkage between the polynucleotides such that the MCNA compound comprises two or more 5′ ends, wherein each polynucleotide codes for hOTC. In some embodiments, a MCNA compound comprises two or more polynucleotides linked via a 3′ end linkage between the polynucleotides such that the MCNA compound comprises two or more 5′ ends, wherein at least one polynucleotide is an encoding polynucleotide that encodes hOTC and at least one polynucleotide acts as a protecting group.

Bridge (w/3′-3′ Linkage)

In some embodiments, a MCNA compound comprises two or more polynucleotides wherein the 3′ ends of each polynucleotide are linked via an oligonucleotide bridge (also “bridging oligonucleotide” or “bridging olio”) comprising a 3′-3′ inverted phosphodiester linkage. In some embodiments, the oligonucleotide bridge comprises modified nucleotides. In some embodiments, the oligonucleotide bridge comprises 2′-O-methyl RNA. In some embodiments, the oligonucleotide bridge comprises DNA. In some embodiments, the oligonucleotide bridge is between 2 and 1000 nucleotides in length. In some embodiments, the oligonucleotide bridge comprises one or more active moieties that are bound to the bridge by covalent links. In some embodiments, an active moiety is a targeting group, peptide, contrast agent, small molecule, protein, DNA and/or RNA. In some embodiments, an active moiety binds a receptor ligand for a cell surface receptor. In some embodiments, the active moiety is one or more tri-antennary GalNac targeting agents.

MCNA Synthesis

In some embodiments, the present invention provides methods of synthesizing MCNA. In some embodiments, the synthesis of MCNA comprises ligating two or more polynucleotides such that the 3′ end of each polynucleotide is ligated to the 5′ end of an oligonucleotide bridge, wherein the oligonucleotide bridge comprises two 5′ ends and an internal 3′-3′ inverted phosphodiester linkage. In some embodiments, the method of synthesizing MCNA comprises the use of oligonucleotide splints complementary to regions of the two or more polynucleotides such that a ligase can join each polynucleotide to a 5′ end of an oligonucleotide bridge. In some embodiments, oligonucleotide splints are complementary to regions of the two or more polynucleotides such that a ligase joins perfect ends of each polynucleotide to a 5′ end of an oligonucleotide bridge. In some embodiments, oligonucleotide splints are complementary to regions of the two or more polynucleotides such that a ligase joins the 3′ end of each polynucleotide to a 5′ end of an oligonucleotide bridge. In some embodiments, an oligonucleotide splint comprises DNA. In some embodiments, a ligase is RNA Ligase. In some embodiments, a ligase is T4 RNA Ligase 1. In some embodiments, a ligase is T4 RNA Ligase 2.

In some embodiments, the molar ratio of polynucleotide to oligonucleotide bridge to oligonucleotide splint when synthesizing MCNA is 2:1:2. In some embodiments, the molar ratio of polynucleotide to oligonucleotide bridge when synthesizing MCNA is 2:1. In some embodiments, the molar ratio of polynucleotide to oligonucleotide splint when synthesizing MCNA is 2:2. In some embodiments, synthesis of MCNA further comprises PEG.

In some embodiments, MCNA can be prepared by splint ligation of the 3′ end of two copies of an RNA to the 5′ ends of a single oligonucleotide containing two 5′ ends and a linked 3′-3′ phosphodiester bond within the sequence. Briefly, a 5′-capped RNA containing a 5′ untranslated region (UTR) and a 3′ UTR flanking an RNA coding sequence is transcribed using T7 RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure and purified. This transcript is then ligated in a single step to a “bridge” oligonucleotide containing a 20 nucleotide (nt) palindromic sequence with a 3′-3′ phosphodiester linkage between the 10^(th) and 11^(th) nt using either (A) T4 RNA ligase 1, (B) T4 RNA ligase 1+PEG 8K, or (C) T4 RNA Ligase 2 and a DNA oligonucleotide “splint” complementary to the 3′-UTR and bridging oligo. To prepare the samples for ligation, the bridging oligo is 5′-end phosphorylated in a reaction containing 50 μM oligo, ATP, 1×PNK Buffer and T4 Polynucleotide Kinase at 37° C. for 1 hour. Phosphorylated bridging oligo is then desalted using a Sephadex G-25 desalting column and hybridized to the transcript and splint in a reaction containing capped RNA transcript, 1× bridging oligo and 2× splint oligo by heating to 75° C. for 5 minutes followed by gradual cooling to room temperature over 5 minutes. An RNA ligation reaction is subsequently prepared to contain a 50% diluted hybridization reaction and (A) 1×RNA ligase Buffer, ATP and T4 RNA ligase 1 (NEB), (B) 1×RNA ligase Buffer, ATP, 10% PEG and T4 RNA ligase 1 (NEB), or (C) 1×T4RNA Ligase 2 Buffer and T4 RNA ligase 2 (NEB). Each is reacted for 90 minutes at 37° C. The completed ligation reaction is then purified using an RNeasy Mini Kit (Qiagen). The purified MCNA product is subsequently treated with DNase Ito remove residual bridge oligonucleotide.

In some embodiments, MCNA can be prepared by splint-independent ligation of the 3′ end of two copies of an RNA to the 5′ ends of a single oligonucleotide containing two 5′ ends and a linked 3′-3′ phosphodiester bond within the sequence.

Untranslated Regions

Typically, mRNA synthesis includes the addition of a “cap” on the 5′ end, and a “tail” on the 3′ end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

In some embodiments, one or more polynucleotides of the MCNA include a 5′ and/or 3′ untranslated region. In some embodiments, a 5′ untranslated region (5′ UTR) includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5′ untranslated region may be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region (3′ UTR) includes one or more of a polyadenylation signal, a binding site for proteins that affect MCNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3′ untranslated region may be between 50 and 500 nucleotides in length or longer. In some embodiments, a 3′ untranslated region may be between 5 and 2,000 nucleotides in length.

Exemplary 3′ and/or 5′ UTR sequences can be derived from nucleic acid molecules that are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense MCNA molecule. For example, a 5′ UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and/or improve the half-life of the polynucleotide. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof to the 3′ end or untranslated region of the polynucleotide (e.g., MCNA) to further stabilize the polynucleotide. Generally, these modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides' resistance to in vivo nuclease digestion.

3′ UTR

In some embodiments, a 3′ UTR comprises a plurality of multi-A segments with spacers in between. In some embodiments, spacers comprise DNA, RNA and/or modified bases. In some embodiments, each of the multi-A segments comprises 8-50 consecutive adenosines. In some embodiments, the plurality of multi-A segments range from 1-100 in number. In some embodiments, the spacers are of varying lengths ranging from 5-100. In some embodiments, a 3′ UTR comprises a pseudoknot structure. A pseudoknot can be defined as an RNA structure minimally composed of two helical segments connected by single stranded regions or loops (Staple, D. W. et al., PLoS Biology, 2005, 3, e213). They are predominantly formed through secondary structures such as hairpin or stem loops and a distal single strand region. In some embodiments, a 3′ UTR comprises a “kissing loop” sequence motif. Broadly defined, a kissing loop can be described as the structure formed when unpaired nucleotides in a stem/hairpin loop of one RNA molecule base pair with unpaired nucleotides of a second stem/hairpin loop of a separate RNA molecule. In some embodiments, a 3′ UTR is not followed with a polyadenylation (poly-A) tail. In some embodiments, a 3′ UTR binds to poly-A binding proteins (PABPs).

In some embodiments, MCNA include a 3′ poly(A) tail structure. In some embodiments, a poly-A tail is 25-5,000 nucleotides in length. A poly-A tail on the 3′ terminus of MCNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3′ poly(C) tail structure. A suitable poly-C tail on the 3′ terminus of MCNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.

Typically, the presence of a “tail” serves to protect the MCNA from exonuclease degradation. The poly A tail is thought to stabilize natural messengers and synthetic sense MCNA. Therefore, in certain embodiments a long poly A tail can be added to an MCNA molecule thus rendering the MCNA more stable. Poly A tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from PCR products. Poly A may also be ligated to the 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).

In some embodiments, one or more polynucleotides of the MCNA includes a 3′ poly(A) tail structure. Typically, the length of the poly-A tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides. In some embodiments, a poly-A tail on the 3′ terminus of MCNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, MCNA include a 3′ poly-C tail structure. A suitable poly-C tail on the 3′ terminus of MCNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.

In some embodiments, the length of the poly-A or poly-C tail is adjusted to control the stability of a modified sense MCNA molecule of the invention and, thus, the transcription of protein that is coded for by one or more of the encoding polynucleotides of the MCNA. For example, since the length of the poly-A tail can influence the half-life of a sense MCNA molecule, the length of the poly-A tail can be adjusted to modify the level of resistance of the MCNA to nucleases and thereby control the time course of polynucleotide expression and/or polypeptide production in a target cell.

5′ UTR

In some embodiments, MCNA include a 5′ cap structure. A 5′ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5′ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

Naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5′-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m⁷G(5′)ppp(5′)N, where N is any nucleoside. In vivo, the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase. The addition of the cap to the 5′ terminal end of RNA occurs immediately after initiation of transcription. The terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5′)ppp(5′)GpNpNp.

One cap for MCNA produced by in vitro transcription is m⁷G(5′)ppp(5′)G, which has been used as the dinucleotide cap in transcription with T7 or SP6 RNA polymerase in vitro to obtain MCNA having a cap structure in their 5′-termini. A method for the in vitro synthesis of capped MCNA employs a pre-formed dinucleotide of the form m⁷G(5′)ppp(5′)G (“m⁷GpppG”) as an initiator of transcription.

To date, a usual form of a synthetic dinucleotide cap used in in vitro translation experiments is the Anti-Reverse Cap Analog (“ARCA”) or modified ARCA, which is generally a modified cap analog in which the 2′ or 3′ OH group is replaced with —OCH₃.

Additional cap analogs include, but are not limited to, a chemical structures selected from the group consisting of m⁷GpppG, m⁷GpppA, m⁷GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m²′⁷GpppG), trimethylated cap analog (e.g., m^(2,2,7)GpppG), dimethylated symmetrical cap analogs (e.g., m⁷Gpppm⁷G), or anti reverse cap analogs (e.g., ARCA; m^(7,2′Ome)GpppG, m^(72′d)GpppG, m^(7,3′Ome)GpppG, m^(7,3′d)GpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs with superior translational properties”, RNA, 9: 1108-1122 (2003)).

In some embodiments, a suitable cap is a 7-methyl guanylate (“m⁷G”) linked via a triphosphate bridge to the 5′-end of the first transcribed nucleotide, resulting in m⁷G(5′)ppp(5′)N, where N is any nucleoside. A preferred embodiment of a m⁷G cap utilized in embodiments of the invention is m⁷G(5′)ppp(5′)G.

In some embodiments, the cap is a Cap0 structure. Cap0 structures lack a 2′-O-methyl residue of the ribose attached to bases 1 and 2. In some embodiments, the cap is a Cap1 structure. Cap1 structures have a 2′-O-methyl residue at base 2. In some embodiments, the cap is a Cap2 structure. Cap2 structures have a 2′-O-methyl residue attached to both bases 2 and 3.

A variety of m⁷G cap analogs are known in the art, many of which are commercially available. These include the m⁷GpppG described above, as well as the ARCA 3′-OCH3 and 2′-OCH3 cap analogs (Jemielity, J. et al., RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodiments of the invention include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)), phosphorothioate cap analogs (described in Grudzien-Nogalska, E., et al., RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylated cap analogs) described in U.S. Pat. Nos. 8,093,367 and 8,304,529, incorporated by reference herein.

Nucleotide Modifications

In some embodiments, MCNA according to the present invention may be synthesized as unmodified or modified nucleic acid. Typically, nucleic acids are modified to enhance stability. Modifications of MCNA can include, for example, modifications of the nucleotides of the MCNA. A modified MCNA according to the invention can thus include, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, MCNA may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as, e.g. 2′-OMe-A, 2′-OMe-G, 2′-OMe-C, 2′-OMe-U, 2′-F-A, 2′-F-G, 2′-F-C, 2′-F-U, LNA-A, LNA-G, LNA-C, LNA-U, N6-methyl-adenosine, 2-thiouridine (2sU), 5-methyl-cytidine (5mC), pseudouridine (ψU), and 1-methyl-pseudouridine, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, .beta.-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g., from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosures of which are incorporated by reference in their entirety.

In some embodiments, MCNA of the of the present invention comprise encoding polynucleotides that comprise one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are selected from the group consisting of 2′-OMe-A, 2′-OMe-G, 2′-OMe-C, 2′-OMe-U, 2′-F-A, 2′-F-G, 2′-F-C, 2′-F-U, LNA-A, LNA-G, LNA-C, LNA-U, N6-methyl-adenosine, 2-thiouridine (2sU), 5-methyl-cytidine (5mC), pseudouridine (ψU), and 1-methyl-pseudouridine. In some embodiments, the modified nucleotides substitute 1-100% of corresponding native bases. In some embodiments, at least 25% of uridines are replaced with 2-thiouridines. In some embodiments, 100% cytidines are replaced with 5-methylcytidines. In some embodiments, modified nucleotides are further modified with a 4′-thio substitution on the ribose ring. In some embodiments, native nucleotides are modified with a 4′-thio substitution on the ribose ring.

In some embodiments, MCNA may contain nucleic acid backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the MCNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5′-O-(1-thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.

In some embodiments, MCNA may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine 5′-triphosphate, 2′-fluoro-2′-deoxyuridine 5′-triphosphate), 2′-deoxy-2′-deamine-oligoribonucleotide (2′-amino-2′-deoxycytidine 5′-triphosphate, 2′-amino-2′-deoxyuridine 5′-triphosphate), 2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyloligoribonucleotide (2′-O-methylcytidine 5′-triphosphate, 2′-methyluridine 5′-triphosphate), 2′-C-alkyloligoribonucleotide, and isomers thereof (2′-aracytidine 5′-triphosphate, 2′-arauridine 5′-triphosphate), or azidotriphosphates (2′-azido-2′-deoxycytidine 5′-triphosphate, 2′-azido-2′-deoxyuridine 5′-triphosphate).

In some embodiments, MCNA may contain modifications of the bases of the nucleotides (base modifications). A modified nucleotide which contains a base modification is also called a base-modified nucleotide. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine riboside 5′-triphosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine 5′-triphosphate, 2-thiouridine 5′-triphosphate, 4-thiouridine 5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate, 5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate, 5-bromouridine 5′-triphosphate, 5-iodocytidine 5′-triphosphate, 5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate, 5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate, 6-azauridine 5′-triphosphate, 6-chloropurine riboside 5′-triphosphate, 7-deazaadenosine 5′-triphosphate, 7-deazaguanosine 5′-triphosphate, 8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate, benzimidazole riboside 5′-triphosphate, N1-methyladenosine 5′-triphosphate, N1-methylguanosine 5′-triphosphate, N6-methyladenosine 5′-triphosphate, 06-methylguanosine 5′-triphosphate, pseudouridine 5′-triphosphate, puromycin 5′-triphosphate or xanthosine 5′-triphosphate. In some embodiments, MCNA comprises modified bases selected from 2′-OMe-A, 2′-OMe-G, 2′-OMe-C, 2′-OMe-U, 2′-F-A, 2′-F-G, 2′-F-C, 2′-F-U, LNA-A, LNA-G, LNA-C, LNA-U, N6-methyl-adenosine, 2-thiouridine (2sU), 5-methyl-cytidine (5mC), pseudouridine (ψU), and 1-methyl-pseudouridine.

Delivery Vehicles

According to the present invention, MCNA as described herein may be delivered as naked polynucleotides or via delivery vehicles. As used herein, the terms “delivery vehicle”, “transfer vehicle”, “nanoparticle” or grammatical equivalent, are used interchangeably.

In some embodiments, MCNA may be delivered via a single delivery vehicle. In some embodiments, MCNA may be delivered via one or more delivery vehicles each of a different composition. According to various embodiments, suitable delivery vehicles include, but are not limited to polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain-block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic polyconjugates), dry powder formulations, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides and other vectorial tags.

Liposomal Delivery Vehicles

In some embodiments, a suitable delivery vehicle is a liposomal delivery vehicle, e.g., a lipid nanoparticle. As used herein, liposomal delivery vehicles, e.g., lipid nanoparticles, are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphophilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, a liposomal delivery vehicle typically serves to transport a desired MCNA to a target cell or tissue.

Cationic Lipids

In some embodiments, liposomes may comprise one or more cationic lipids. As used herein, the phrase “cationic lipid” refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available. Particularly suitable cationic lipids for use in the compositions and methods of the invention include those described in international patent publications WO 2010/053572 (and particularly, CI 2-200 described at paragraph [00225]) and WO 2012/170930, both of which are incorporated herein by reference. In certain embodiments, the compositions and methods of the invention employ a lipid nanoparticles comprising an ionizable cationic lipid described in U.S. provisional patent application 61/617,468, filed Mar. 29, 2012 (incorporated herein by reference), such as, e.g, (15Z, 18Z)—N,N-dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z, 18Z)—N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)—N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-5, 15, 18-trien-1-amine (HGT5002).

In some embodiments, provided liposomes include a cationic lipid described in WO 2013/063468 and in U.S. provisional application entitled “Lipid Formulations for Delivery of Messenger RNA” filed concurrently with the present application on even date, both of which are incorporated by reference herein.

In some embodiments, a cationic lipid comprises a compound of formula I-c1-a:

or a pharmaceutically acceptable salt thereof, wherein:

each R² independently is hydrogen or C₁₋₃ alkyl;

each q independently is 2 to 6;

each R′ independently is hydrogen or C₁₋₃ alkyl;

and each R^(L) independently is C8-12 alkyl.

In some embodiments, each R² independently is hydrogen, methyl or ethyl. In some embodiments, each R² independently is hydrogen or methyl. In some embodiments, each R² is hydrogen.

In some embodiments, each q independently is 3 to 6. In some embodiments, each q independently is 3 to 5. In some embodiments, each q is 4.

In some embodiments, each R′ independently is hydrogen, methyl or ethyl. In some embodiments, each R′ independently is hydrogen or methyl. In some embodiments, each R′ independently is hydrogen.

In some embodiments, each R^(L) independently is C8-12 alkyl. In some embodiments, each R^(L) independently is n-C₅₋₁₂ alkyl. In some embodiments, each R^(L) independently is C₉₋₁₁ alkyl. In some embodiments, each R^(L) independently is n-C₉₋₁₁ alkyl. In some embodiments, each R^(L) independently is C₁₀ alkyl. In some embodiments, each R^(L) independently is n-C₁₀ alkyl.

In some embodiments, each R² independently is hydrogen or methyl; each q independently is 3 to 5; each R′ independently is hydrogen or methyl; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² is hydrogen; each q independently is 3 to 5; each R′ is hydrogen; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² is hydrogen; each q is 4; each R′ is hydrogen; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, a cationic lipid comprises a compound of formula I-g:

or a pharmaceutically acceptable salt thereof, wherein each R^(L) independently is C₈₋₁₂ alkyl. In some embodiments, each R^(L) independently is n-C₈₋₁₂ alkyl. In some embodiments, each R^(L) independently is C₉₋₁₁ alkyl. In some embodiments, each R^(L) independently is n-C₉₋₁₁ alkyl. In some embodiments, each R^(L) independently is C₁₀ alkyl. In some embodiments, each R^(L) is n-C₁₀ alkyl.

In particular embodiments, provided liposomes include a cationic lipid cKK-E12, or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione). The structure of cKK-E12 is shown below:

Additional exemplary cationic lipids include those of formula I:

and pharmaceutically acceptable salts thereof, wherein,

(see, e.g., Fenton, Owen S., et al. “Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery.” Advanced materials (2016)).

In some embodiments, the one or more cationic lipids may be N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or “DOTMA” (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or can be combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine or “DOPE” or other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells. Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or “DOGS,” 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium or “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S. Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propane or “DODAP”, 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”.

Additional exemplary cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”, 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”, 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”, 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”, N-dioleyl-N,N-dimethylammonium chloride or “DODAC”, N,N-distearyl-N,N-dimethylarnrnonium bromide or “DDAB”, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide or “DMRIE”, 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane or “CLinDMA”, 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy 1-1-(cis,cis-9′,1-2′-octadecadienoxy)propane or “CpLinDMA”, N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”, 1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”, 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”, 1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”, 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”, 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin-DMA”, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or “DLin-K-XTC2-DMA”, and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech. 28: 172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). In some embodiments, one or more of the cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.

In some embodiments, the one or more cationic lipids may be chosen from XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide), DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889, the teachings of which are incorporated herein by reference in their entirety), ICE (WO 2011/068810, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U.S. Provisional Patent Application No. 61/617,468, the teachings of which are incorporated herein by reference in their entirety) or HGT5001 (cis or trans) (Provisional Patent Application No. 61/617,468), aminoalcohol lipidoids such as those disclosed in WO2010/053572, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids” J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al. “Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech. 2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materials for low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869).

In some embodiments, the percentage of cationic lipid in a liposome may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. In some embodiments, cationic lipid(s) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by weight. In some embodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30%, about 35%, about 40%, about 45%, or about 50% of the liposome by molar ratio.

Non-Cationic/Helper Lipids

In some embodiments, provided liposomes contain one or more non-cationic (“helper”) lipids. As used herein, the phrase “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase “anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected H, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof.

In some embodiments, such non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids. In some embodiments, the non-cationic lipid may comprise a molar ratio of about 5% to about 90%, or about 10% to about 70% of the total lipid present in a liposome. In some embodiments, a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

Cholesterol-Based Lipids

In some embodiments, provided liposomes comprise one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipid may comprise a molar ration of about 2% to about 30%, or about 5% to about 20% of the total lipid present in a liposome. In some embodiments, The percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than 5, %, 10%, greater than 20%, greater than 30%, or greater than 40%.

PEGylated Lipids

In some embodiments, provided liposomes comprise one or more PEGylated lipids. For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention in combination with one or more of the cationic and, in some embodiments, other lipids together which comprise the liposome. Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).

In some embodiments, particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C₁₄ or C₁₈). The PEG-modified phospholipid and derivitized lipids of the present invention may comprise a molar ratio from about 0% to about 15%, about 0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposome.

According to various embodiments, the selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the MCNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus the molar ratios may be adjusted accordingly.

Formation of Liposomes

The liposomal transfer vehicles for use in the compositions of the invention can be prepared by various techniques which are presently known in the art. The liposomes for use in provided compositions can be prepared by various techniques which are presently known in the art. For example, multilamellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then added to the vessel with a vortexing motion which results in the formation of MLVs. Unilamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

In certain embodiments, provided compositions comprise a liposome wherein the MCNA is associated on both the surface of the liposome and encapsulated within the same liposome. For example, during preparation of the compositions of the present invention, cationic liposomes may associate with the MCNA through electrostatic interactions. For example, during preparation of the compositions of the present invention, cationic liposomes may associate with the MCNA through electrostatic interactions.

In some embodiments, the compositions and methods of the invention comprise MCNA encapsulated in a liposome. In some embodiments, the one or more MCNA species may be encapsulated in the same liposome. In some embodiments, the one or more MCNA species may be encapsulated in different liposomes. In some embodiments, the MCNA is encapsulated in one or more liposomes, which differ in their lipid composition, molar ratio of lipid components, size, charge (Zeta potential), targeting ligands and/or combinations thereof. In some embodiments, the one or more liposome may have a different composition of cationic lipids, neutral lipid, PEG-modified lipid and/or combinations thereof. In some embodiments the one or more liposomes may have a different molar ratio of cationic lipid, neutral lipid, cholesterol and PEG-modified lipid used to create the liposome.

The process of incorporation of a desired MCNA into a liposome is often referred to as “loading”. Exemplary methods are described in Lasic, et al., FEBS Lett., 312: 255-258, 1992, which is incorporated herein by reference. The liposome-incorporated nucleic acids may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane. The incorporation of a nucleic acid into liposomes is also referred to herein as “encapsulation” wherein the nucleic acid is entirely contained within the interior space of the liposome. The purpose of incorporating a MCNA into a transfer vehicle, such as a liposome, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and/or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some embodiments, a suitable delivery vehicle is capable of enhancing the stability of the MCNA contained therein and/or facilitate the delivery of MCNA to the target cell or tissue.

Liposome Size

Suitable liposomes in accordance with the present invention may be made in various sizes. In some embodiments, provided liposomes may be made smaller than previously known mRNA encapsulating liposomes. In some embodiments, decreased size of liposomes is associated with more efficient delivery of MCNA. Selection of an appropriate liposome size may take into consideration the site of the target cell or tissue and to some extent the application for which the liposome is being made.

In some embodiments, an appropriate size of lipo some is selected to facilitate systemic distribution of polypeptide encoded by the MCNA. In some embodiments, it may be desirable to limit transfection of the MCNA to certain cells or tissues. For example, to target hepatocytes a liposome may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; in such cases the liposome could readily penetrate such endothelial fenestrations to reach the target hepatocytes.

Alternatively or additionally, a liposome may be sized such that the dimensions of the liposome are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues. For example, a liposome may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the liposomes to hepatocytes.

In some embodiments, the size of a liposome is determined by the length of the largest diameter of the liposome particle. In some embodiments, a suitable liposome has a size no greater than about 250 nm (e.g., no greater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, or 50 nm). In some embodiments, a suitable liposome has a size ranging from about 10-250 nm (e.g., ranging from about 10-225 nm, 10-200 nm, 10-175 nm, 10-150 nm, 10-125 nm, 10-100 nm, 10-75 nm, or 10-50 nm). In some embodiments, a suitable liposome has a size ranging from about 100-250 nm (e.g., ranging from about 100-225 nm, 100-200 nm, 100-175 nm, 100-150 nm). In some embodiments, a suitable liposome has a size ranging from about 10-100 nm (e.g., ranging from about 10-90 nm, 10-80 nm, 10-70 nm, 10-60 nm, or 10-50 nm). In a particular embodiment, a suitable liposome has a size less than about 100 nm.

A variety of alternative methods known in the art are available for sizing of a population of liposomes. One such sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small ULV less than about 0.05 microns in diameter. Homogenization is another method that relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, MLV are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. The size of the liposomes may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), incorporated herein by reference. Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.

Polymers

In some embodiments, a suitable delivery vehicle is formulated using a polymer as a carrier, alone or in combination with other carriers including various lipids described herein. Thus, in some embodiments, liposomal delivery vehicles, as used herein, also encompass polymer containing nanoparticles. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine (PEI). When PEI is present, it may be branched PEI of a molecular weight ranging from 10 to 40 kDa, e.g., 25 kDa branched PEI (Sigma #408727).

A suitable liposome for the present invention may include one or more of any of the cationic lipids, non-cationic lipids, cholesterol lipids, PEGylated lipids and/or polymers described herein at various ratios. As non-limiting examples, a suitable liposome formulation may include a combination selected from cKK-E12, DOPE, cholesterol and DMG-PEG2K; C₁₂-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, cholesterol and DMG-PEG2K.

In various embodiments, cationic lipids (e.g., cKK-E12, C12-200, ICE, and/or HGT4003) constitute about 30-60% (e.g., about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by molar ratio. In some embodiments, the percentage of cationic lipids (e.g., cKK-E12, C12-200, ICE, and/or HGT4003) is or greater than about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the liposome by molar ratio.

In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEGylated lipid(s) may be between about 30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEGylated lipid(s) is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEGylated lipid(s) is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEGylated lipid(s) is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEGylated lipid(s) is approximately 50:25:20:5.

Pharmaceutical Compositions

To facilitate expression of MCNA in vivo, delivery vehicles such as liposomes can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.

In some embodiments, a composition comprises MCNA encapsulated or complexed with a delivery vehicle. In some embodiments, the delivery vehicle is selected from the group consisting of liposomes, lipid nanoparticles, solid-lipid nanoparticles, polymers, viruses, sol-gels, and nanogels.

Provided liposomally-encapsulated or liposomally-associated MCNA, and compositions containing the same, may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art. The “effective amount” for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art. For example, a suitable amount and dosing regimen is one that causes at least transient protein (e.g., enzyme) production.

The present invention provides methods of delivering MCNA for in vivo protein production, comprising administering MCNA to a subject in need of delivery. In some embodiments, MCNA is administered via a route of delivery selected from the group consisting of intravenous delivery, subcutaneous delivery, oral delivery, subdermal delivery, ocular delivery, intratracheal injection pulmonary delivery (e.g. nebulization), intramuscular delivery, intrathecal delivery, or intraarticular delivery.

Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal. In particular embodiments, the intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle and cardiac muscle. In some embodiments the administration results in delivery of the MCNA to a muscle cell. In some embodiments the administration results in delivery of the MCNA to a hepatocyte (i.e., liver cell). In a particular embodiment, the intramuscular administration results in delivery of the MCNA to a muscle cell.

Alternatively or additionally, liposomally-encapsulated MCNA and compositions of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a targeted tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted. For example, aerosols containing compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection. Formulations containing provided compositions complexed with therapeutic molecules or ligands can even be surgically administered, for example in association with a polymer or other structure or substance that can allow the compositions to diffuse from the site of implantation to surrounding cells. Alternatively, they can be applied surgically without the use of polymers or supports.

Provided methods of the present invention contemplate single as well as multiple administrations of a therapeutically effective amount of the therapeutic agents (e.g., MCNA) described herein. Therapeutic agents can be administered at regular intervals, depending on the nature, severity and extent of the subject's condition. In some embodiments, a therapeutically effective amount of the therapeutic agents (e.g., MCNA) of the present invention may be administered intrathecally periodically at regular intervals (e.g., once every year, once every six months, once every five months, once every three months, bimonthly (once every two months), monthly (once every month), biweekly (once every two weeks), twice a month, once every 30 days, once every 28 days, once every 14 days, once every 10 days, once every 7 days, weekly, twice a week, daily or continuously).

In some embodiments, provided liposomes and/or compositions are formulated such that they are suitable for extended-release of the MCNA contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. For example, in one embodiment, the compositions of the present invention are administered to a subject twice a day, daily or every other day. In a preferred embodiment, the compositions of the present invention are administered to a subject twice a week, once a week, once every 7 days, once every 10 days, once every 14 days, once every 28 days, once every 30 days, once every two weeks, once every three weeks, or more preferably once every four weeks, once a month, twice a month, once every six weeks, once every eight weeks, once every other month, once every three months, once every four months, once every six months, once every eight months, once every nine months or annually. Also contemplated are compositions and liposomes which are formulated for depot administration (e.g., intramuscularly, subcutaneously, intravitreally) to either deliver or release MCNA over extended periods of time. Preferably, the extended-release means employed are combined with modifications made to the MCNA to enhance stability.

As used herein, the term “therapeutically effective amount” is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present invention. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., treating, modulating, curing, preventing and/or ameliorating a disease or disorder). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and/or prophylactic effect. Generally, the amount of a therapeutic agent (e.g., MCNA) administered to a subject in need thereof will depend upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject. One of ordinary skill in the art will be readily able to determine appropriate dosages depending on these and other related factors. In addition, both objective and subjective assays may optionally be employed to identify optimal dosage ranges.

A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific protein employed; the duration of the treatment; and like factors as is well known in the medical arts.

In some embodiments, the therapeutically effective dose ranges from about 0.005 mg/kg body weight to 500 mg/kg body weight, e.g., from about 0.005 mg/kg body weight to 400 mg/kg body weight, from about 0.005 mg/kg body weight to 300 mg/kg body weight, from about 0.005 mg/kg body weight to 200 mg/kg body weight, from about 0.005 mg/kg body weight to 100 mg/kg body weight, from about 0.005 mg/kg body weight to 90 mg/kg body weight, from about 0.005 mg/kg body weight to 80 mg/kg body weight, from about 0.005 mg/kg body weight to 70 mg/kg body weight, from about 0.005 mg/kg body weight to 60 mg/kg body weight, from about 0.005 mg/kg body weight to 50 mg/kg body weight, from about 0.005 mg/kg body weight to 40 mg/kg body weight, from about 0.005 mg/kg body weight to 30 mg/kg body weight, from about 0.005 mg/kg body weight to 25 mg/kg body weight, from about 0.005 mg/kg body weight to 20 mg/kg body weight, from about 0.005 mg/kg body weight to 15 mg/kg body weight, from about 0.005 mg/kg body weight to 10 mg/kg body weight.

In some embodiments, the therapeutically effective dose is greater than about 0.1 mg/kg body weight, greater than about 0.5 mg/kg body weight, greater than about 1.0 mg/kg body weight, greater than about 3 mg/kg body weight, greater than about 5 mg/kg body weight, greater than about 10 mg/kg body weight, greater than about 15 mg/kg body weight, greater than about 20 mg/kg body weight, greater than about 30 mg/kg body weight, greater than about 40 mg/kg body weight, greater than about 50 mg/kg body weight, greater than about 60 mg/kg body weight, greater than about 70 mg/kg body weight, greater than about 80 mg/kg body weight, greater than about 90 mg/kg body weight, greater than about 100 mg/kg body weight, greater than about 150 mg/kg body weight, greater than about 200 mg/kg body weight, greater than about 250 mg/kg body weight, greater than about 300 mg/kg body weight, greater than about 350 mg/kg body weight, greater than about 400 mg/kg body weight, greater than about 450 mg/kg body weight, greater than about 500 mg/kg body weight. In a particular embodiment, the therapeutically effective dose is 1.0 mg/kg. In some embodiments, the therapeutically effective dose of 1.0 mg/kg is administered intramuscularly or intravenously.

Also contemplated herein are lyophilized pharmaceutical compositions comprising one or more of the liposomes disclosed herein and related methods for the use of such compositions as disclosed for example, in U.S. Provisional Application No. 61/494,882, filed Jun. 8, 2011, the teachings of which are incorporated herein by reference in their entirety. For example, lyophilized pharmaceutical compositions according to the invention may be reconstituted prior to administration or can be reconstituted in vivo. For example, a lyophilized pharmaceutical composition can be formulated in an appropriate dosage form (e.g., an intradermal dosage form such as a disk, rod or membrane) and administered such that the dosage form is rehydrated over time in vivo by the individual's bodily fluids.

Provided liposomes and compositions may be administered to any desired tissue. In some embodiments, the MCNA delivered by provided liposomes or compositions is expressed in the tissue in which the liposomes and/or compositions were administered. In some embodiments, the MCNA delivered is expressed in a tissue different from the tissue in which the liposomes and/or compositions were administered. Exemplary tissues in which delivered MCNA may be delivered and/or expressed include, but are not limited to the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid.

In some embodiments, administering the provided composition results in an increased MCNA expression level in a biological sample from a subject as compared to a baseline expression level before treatment. Typically, the baseline level is measured immediately before treatment. Biological samples include, for example, whole blood, serum, plasma, urine and tissue samples (e.g., muscle, liver, skin fibroblasts). In some embodiments, administering the provided composition results in an increased MCNA expression level by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to the baseline level immediately before treatment. In some embodiments, administering the provided composition results in an increased MCNA expression level as compared to a MCNA expression level in subjects who are not treated

According to various embodiments, the timing of expression of delivered MCNA can be tuned to suit a particular medical need. In some embodiments, the expression of the protein encoded by delivered MCNA is detectable 1, 2, 3, 6, 12, 24, 48, 72, and/or 96 hours after administration of provided liposomes and/or compositions. In some embodiments, the expression of the protein encoded by delivered MCNA is detectable 1 week, two weeks, and/or 1 month after administration.

EXAMPLES

While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same.

Example 1. Exemplary Synthesis of Multimeric Coding Nucleic Acid (MCNA)

This example provides exemplary schemes for synthesizing the MCNA described in this application, for effective delivery and expression of MCNA encoding therapeutic proteins in vivo.

Synthesis of MCNA was attempted by ligating a synthetic oligonucleotide containing a 3′-3′ phosphodiester bond to multiple polynucleotides using a complementary DNA splint. Several different T4 RNA ligases were tested for the ability to ligate a synthetic oligonucleotide containing a 3′-3′ phosphodiester bond to multiple polynucleotides using a complementary DNA splint. The first RNA ligase (“RNA Ligase 1”) was a “single-strand” RNA ligase that ligated single RNA strands, double RNA strands and double RNA strands designed to implement a single strand overhang. The second RNA ligase (“RNA Ligase 2”) was a “double-stranded” RNA ligase that ligated nicks in RNA bound to a complementary oligonucleotide. Both RNA Ligase 1 and RNA Ligase 2 required phosphorylated 5′ ends of the oligonucleotide bridge to proceed with adenylation for the ligation reaction.

As a non-limiting example, Erythropoietin (EPO) mRNA was ligated to a bridging oligo containing a 3′-3′ phosphodiester bond using a complementary DNA splint. Examples of a bridging oligonucleotide that contains a 3′-3′ phosphodiester bond and DNA splints are described below. The exemplary sequence for EPO used in the examples herein are listed below.

Erythropoietin (EPO) mRNA (including 5′ and 3′ UTR): (SEQ ID NO: 1) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACC GGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG UGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGGGUGCACGAAUGUCCUGCCU GGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCUCUGGGCCUCCCAGUCCUGGG CGCCCCACCACGCCUCAUCUGUGACAGCCGAGUCCUGGAGAGGUACCUCUUGGAG GCCAAGGAGGCCGAGAAUAUCACGACGGGCUGUGCUGAACACUGCAGCUUGAAU GAGAAUAUCACUGUCCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUG GAGGUCGGGCAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAG CUGUCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCU GCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUU CGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAGCUG CUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGAGUCUACUC CAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGCCUGCAGGACAGG GGACAGAUGACGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCU GGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC AAGCU Erythropoietin (EPO) mRNA (including 5′ and 3′ UTR with 200 A poly(A) Tail): (SEQ ID NO: 2) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACC GGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG UGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGGGUGCACGAAUGUCCUGCCU GGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCUCUGGGCCUCCCAGUCCUGGG CGCCCCACCACGCCUCAUCUGUGACAGCCGAGUCCUGGAGAGGUACCUCUUGGAG GCCAAGGAGGCCGAGAAUAUCACGACGGGCUGUGCUGAACACUGCAGCUUGAAU GAGAAUAUCACUGUCCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUG GAGGUCGGGCAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAG CUGUCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCU GCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUU CGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAGCUG CUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGAGUCUACUC CAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGCCUGCAGGACAGG GGACAGAUGACGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCU GGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC AAGCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Erythropoietin (EPO) mRNA (including 5′ and 3′ UTR with internal 65A poly(A) region in 3′ UTR): (SEQ ID NO: 3) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACC GGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG UGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGGGUGCACGAAUGUCCUGCCU GGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCUCUGGGCCUCCCAGUCCUGGG CGCCCCACCACGCCUCAUCUGUGACAGCCGAGUCCUGGAGAGGUACCUCUUGGAG GCCAAGGAGGCCGAGAAUAUCACGACGGGCUGUGCUGAACACUGCAGCUUGAAU GAGAAUAUCACUGUCCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUG GAGGUCGGGCAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAG CUGUCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCU GCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUU CGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAGCUG CUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGAGUCUACUC CAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGCCUGCAGGACAGG GGACAGAUGACGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCU GGAAGUUGCCACUCCAGUGCCCACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCCUUGUCCUAAUAAAAU UAAGUUGCAUCAAGCU Erythropoietin (EPO) mRNA (including 5′ and 3′ UTR with multiple short internal poly(A) regions in 3′ UTR): (SEQ ID NO: 4) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACC GGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG UGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGGGUGCACGAAUGUCCUGCCU GGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCUCUGGGCCUCCCAGUCCUGGG CGCCCCACCACGCCUCAUCUGUGACAGCCGAGUCCUGGAGAGGUACCUCUUGGAG GCCAAGGAGGCCGAGAAUAUCACGACGGGCUGUGCUGAACACUGCAGCUUGAAU GAGAAUAUCACUGUCCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUG GAGGUCGGGCAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAG CUGUCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCU GCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUU CGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAGCUG CUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGAGUCUACUC CAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGCCUGCAGGACAGG GGACAGAUGACGGGUGGCAAAAAAAAAAAAAAAUCCCUGUGACCCCUCCCCAAAA AAAAAAAAAAAAGUGCCUCUCCUGGCCCUGGAAAAAAAAAAAAAAAGUUGCCAC UCCAGUGCCCACCAAAAAAAAAAAAAAAGCCUUGUCCUAAUAAAAUUAAGUUGC AUCAAGCU Bridging Oligonucleotide 1: (SEQ ID NO: 5) 5′-CGA CUC UCG G-3′-PO₄-3′-G GCU CUC AGC-5′ The bases included in SEQ ID NO: 5 are 2′-O-methyl RNA and the 3′-3′ bridge comprises PO₄ Bridging Oligonucleotide 2: (SEQ ID NO: 6) 5′-AAAAAAAAAA-3′-PO 4-3′-AAAAAAAAAA-5′ Bridging Oligonucleotide 3: (SEQ ID NO: 7) 5′-AAA-3′-PO 4-3′-AAA-5′ Bridging Oligonucleotide 4: (SEQ ID NO: 8) 5′-A-3′-PO 4-3′-A-5′ Splint Oligonucleotide 1: (SEQ ID NO: 9) 5′-CCG AGA GTC GAG CTT GAT GCA ACT TAA TTT TAT TAG G-3′ Splint Oligonucleotide 2: (SEQ ID NO: 10) 5′-CCG AGA GTG ATG CAA CTT AAT TTT ATT AGG-3′ Splint Oligonucleotide 3: (SEQ ID NO: 11) 5′-TTT TTT TTT TAG CTT GAT GCA ACT TAA TTT TAT TAG G-3′ Splint Oligonucleotide 4: (SEQ ID NO: 12) 5′-CCG AGA GTC GTT TTT TTT TTT TTT TTT TTT-3′ Splint Oligonucleotide 5: (SEQ ID NO: 13) 3′-G GAT TAT TTT AAT TCA ACG TAG TTC GAG CTG AGA GCC-5′-PO₄-5′-CCG AGA GTC GAG CTT GAT GCA ACT TAA TTT TAT TAG G-3′ Splint Oligonucleotide 6: (SEQ ID NO: 14) 3′-GGA TTA TTT TAA TTC AAC GTA GTG AGA GCC-5′-PO₄-5′-CCG AGA GTG ATG CAA CTT AAT TTT ATT AGG-3′ Splint Oligonucleotide 7: (SEQ ID NO: 15) 3′-G GAT TAT TTT AAT TCA ACG TAG TTC GAT TTT TTT TTT-5′-PO₄-5′-TTT TTT TTT TAG CTT GAT GCA ACT TAA TTT TAT TAG G-3′ Splint Oligonucleotide 8: (SEQ ID NO: 16) 3′-TTT TTT TTT TTT TTT TTT TTG CTG AGA GCC-5′-PO₄-5′-CCG AGA GTC GTT TTT TTT TTT TTT TTT TTT-3′

EPO MCNA #1 (No Poly a Tail)

MCNA 1 (SEQ ID NO: 17) was prepared by splint ligation of the 3′ end of two copies of an RNA encoding the human Erythropoietin (hEPO) protein to the 5′ ends of a single oligonucleotide containing two 5′ ends and a linked 3′-3′ phosphodiester bond within the sequence. Briefly, a 5′-capped RNA containing a 5′ untranslated region (UTR) and a 3′ UTR flanking an RNA sequence encoding hEPO was transcribed using T7 RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure, and purified. This hEPO transcript was then ligated in a single step to a 2′-hydroxymethylated RNA (OMeRNA) “bridging” oligonucleotide containing a 20 nucleotide (nt) palindromic sequence with a 3′-3′ phosphodiester linkage between the 10^(th) and 11^(th) nt (bridging oligo 1 (SEQ ID NO: 5); 5′-CGA CUC UCG G-3′-3′-G GCU CUC AGC-5′, bold bases OMeRNA) using either (A) T4 RNA ligase 1+PEG 8K, (B) T4 RNA ligase 1 or (C) T4 RNA Ligase 2 and a DNA oligonucleotide “splint” complementary to the 3′-UTR and bridging oligo 1 (splint oligo 1 (SEQ ID NO: 9); 5′ CCG AGA GTC GAG CTT GAT GCA ACT TAA TTT TAT TAG G 3′; all bases DNA). Alternatively, MCNA was prepared using splint oligonucleotide 5 (SEQ ID NO: 13), a palindromic sequence containing 2 copies of oligo 2 connected with a 5′-5′ phosphodiester bond. To prepare the samples for ligation, bridging oligo 1 was 5′-end phosphorylated in a reaction containing 50 μM bridging oligo 1, 1 mM ATP, 1×PNK Buffer (NEB; 70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT pH 7.6 at 25° C.) and 0.5 U/μL T4 Polynucleotide Kinase (NEB) at 37° C. for 1 h. Phosphorylated bridging oligo 1 was then desalted using a Sephadex G-25 desalting column (Princeton Separations) and hybridized to the transcript and splint in a reaction containing 3.2 μM capped hEPO transcript, 1.5 μM bridging oligo 1 and 3 μM splint oligo 1 (or 1.5 uM splint oligo 5) by heating to 75° C. for 5 min followed by gradual cooling to room temperature over 5 min. An RNA ligation reaction was subsequently prepared to contain a 50% diluted hybridization reaction and (A) 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT pH 7.5 at 25° C.), 1 mM ATP and 1 U/μL T4 RNA ligase 1 (NEB), (B) 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT pH 7.5 at 25° C.), 1 mM ATP, 10% PEG and 1 U/μL T4 RNA ligase 1 (NEB) or (C) 1×T4RNA Ligase 2 Buffer (NEB; 50 mM Tris-HCl, 2 mM MgCl₂, 1 mM DTT, 400 μM ATP at pH 7.5 at 25° C.) and 1 U/μL T4 RNA ligase 2 (NEB). Each was reacted for 90 minutes at 37° C. The completed ligation reaction was then purified using an RNeasy Mini Kit (Qiagen). A portion of the purified MCNA 1 product was subsequently treated with DNase I to remove residual bridge oligonucleotide to prevent potential endogenous RNase H cleavage of PCNA 1 in cells.

Alternatively, MCNA 1 (SEQ ID NO: 17) was prepared by splint ligation of the 3′ end of two copies of an RNA encoding the human Erythropoietin (hEPO) protein to the 5′ ends of a single oligonucleotide containing two 5′ ends and a linked 3′-3′ phosphodiester bond within the sequence. Briefly, a 5′-capped RNA containing a 5′-untranslated region (UTR) and a 3′ UTR flanking an RNA sequence encoding hEPO was transcribed using T7 RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure and purified. This hEPO transcript was then ligated in a single step to a 2′-hydroxymethylated RNA (OMeRNA) “bridging” oligonucleotide containing a 20 nucleotide (nt) palindromic sequence with a 3′-3′ phosphodiester linkage between the 10^(th) and 11^(th) nt (bridging oligo 1 (SEQ ID NO: 5); 5′-CGA CUC UCG G-3′-3′-G GCU CUC AGC-5′, bold bases OMeRNA) using either (A) T4 RNA ligase 1+PEG 8K, (B) T4 RNA ligase 1 or (C) T4 RNA Ligase 2 and a DNA oligonucleotide “splint” complementary to the 3′-UTR and bridging oligo 1 (splint oligo 1 (SEQ ID NO: 9); 5′ CCG AGA GTC GAG CTT GAT GCA ACT TAA TTT TAT TAG G 3′; all bases DNA). Alternatively, MCNA was prepared using splint oligonucleotide 6 (SEQ ID NO: 14), and a palindromic sequence containing 2 copies of oligo 2 connected with a 5′-5′ phosphodiester bond. To prepare the samples for ligation, bridging oligo 1 was 5′-end phosphorylated in a reaction containing 50 μM bridging oligo 1, 1 mM ATP, 1×PNK Buffer (NEB; 70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT pH 7.6 at 25° C.) and 0.5 U/μL T4 Polynucleotide Kinase (NEB) at 37° C. for 1 hour. Phosphorylated bridging oligo 1 was then desalted using a Sephadex G-25 desalting column (Princeton Separations) and hybridized to the transcript and splint in a reaction containing 3.2 μM capped hEPO transcript, 1.5 μM bridging oligo 1 and 3 μM splint oligo 1 (or 1.5 uM splint oligo 6) by heating to 75° C. for 5 minutes followed by gradual cooling to room temperature over 5 minutes. An RNA ligation reaction was subsequently prepared to contain a 50% diluted hybridization reaction and (A) 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT pH 7.5 at 25° C.), 1 mM ATP and 1 U/μL T4 RNA ligase 1 (NEB), (B) 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT pH 7.5 at 25° C.), 1 mM ATP, 10% PEG and 1 U/μL T4 RNA ligase 1 (NEB) or (C) 1×T4RNA Ligase 2 Buffer (NEB; 50 mM Tris-HCl, 2 mM MgCl₂, 1 mM DTT, 400 μM ATP pH 7.5 at 25° C.) and 1 U/μL T4 RNA ligase 2 (NEB). Each was reacted for 90 minutes 37° C. The completed ligation reaction was then purified using an RNeasy Mini Kit (Qiagen). A portion of the purified MCNA 1 product was subsequently treated with DNase I to remove residual bridge oligonucleotide to prevent potential endogenous RNase H cleavage of PCNA 1 in cells.

MCNA 1 (No Poly(A) Tail Sequence): (SEQ ID NO: 17) 5′- GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAG ACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGC GGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGGGUGC ACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCU CUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGACAGCCG AGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUAUCACGA CGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGUCCCAGAC ACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGGCAGCAGGC CGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUGUCCUGCGGG GCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCUGCAGCUG CAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUUCG GGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAG CUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGA GUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGC CUGCAGGACAGGGGACAGAUGACGGGUGGCAUCCCUGUGACCCCUCCCCA GUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGU CCUAAUAAAAUUAAGUUGCAUCAAGCU

UCGAACUACGUUGAAUUAAAAUAAUCCUGUUCCGACCACC CGUGACCUCACCGUUGAAGGUCCCGGUCCUCUCCGUGACCCCUCCCCAGU GUCCCUACGGUGGGCAGUAGACAGGGGACAGGACGUCCGGAGGGGACACA UGUCGAAGUCGAAAGGGGCCUCCUUUAACCUCAUCUGAGCCUUCUCAAAC GCCUUUCACAGUCGUCACUAACAAGCCUCACCUCGUCGACUCCGGCGUAG ACCUCCCCUCUACCGAAGGAAGACCCGAGGGUCUCGGGCUUCGUCUCACC ACUCCGACGCUUCCGGUGACUGCCGAAAUAGGUGUACGUCGACGUCCCCG AGGGUGCCGACCCUUCUCAACUGGUUGUCCCGGACCGGGGCGUCCUGUCG AAGGCUGUCGUCCCGGUCCGGGACGGUCUGAAGAUGCCGGACGACGGGCU GGAGGUAGGAGAAGGUCCGUAUCUUUAAUUGAAACCACAGACCCUGUCAC UAUAAGAGUAAGUUCGACGUCACAAGUCGUGUCGGGCAGCACUAUAAGAG CCGGAGGAACCGGAGGUUCUCCAUGGAGAGGUCCUGAGCCGACAGUGUCU ACUCCGCACCACCCCGCGGGUCCUGACCCUCCGGGUCUCCCUCGCUGUCG UCCCUGUCCUCUUCGGUGUCGGUCCGUCCUGUAAGCACGUGGGGGUAGCA CAGUUCCUGCCACUCAGUGAGAACCGUGCCCCUUAGGCGCAAGGUUACGU GGCAAGGGCCGGCGCCUCCGACCUAGCCAGGGCCACAGAAGAUACCUCCA GUUUUGUCGCACCUACCGCAGAGGUCCGCUAGACAGG-5′

EPO MCNA #2

MCNA 2 (SEQ ID NO: 18) was prepared by splint ligation of the 3′ end of two copies of an RNA encoding the human Erythropoietin (hEPO) protein to the 5′ ends of a single oligonucleotide containing two 5′ ends and a linked 3′-3′ phosphodiester bond within the sequence. Briefly, a 5′-capped RNA containing a 5′-untranslated region (UTR) and a 3′ UTR flanking an RNA sequence encoding hEPO was transcribed using T7 RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure and purified. This hEPO transcript was then ligated in a single step to an RNA “bridging” oligonucleotide containing a 20 nucleotide (nt) palindromic sequence with a 3′-3′ phosphodiester linkage between the 10^(th) and 11^(th) nt (bridging oligo 2 (SEQ ID NO: 6); 5′-AAA AAA AAA A-3′-3′-A AAA AAA AAA-5′, underlined bases RNA) using T4 RNA ligase 1+PEG 8K and a DNA oligonucleotide “splint” complementary to the 3′-UTR and bridging oligo 2 (splint oligo 3 (SEQ ID NO: 11); 5′ TTT TTT TTT TAG CTT GAT GCA ACT TAA TTT TAT TAG G 3′; all bases DNA). Alternatively, MCNA was prepared using splint oligo 7 (SEQ ID NO: 15), a palindromic sequence containing 2 copies of splint oligo 7 connected with a 5′-5′ phosphodiester bond. To prepare the samples for ligation, bridging oligo 2 was 5′-end phosphorylated in a reaction containing 50 μM oligo 3, 1 mM ATP, 1×PNK Buffer (NEB; 70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT, pH 7.6 at 25° C.) and 0.5 U/μL T4 Polynucleotide Kinase (NEB) at 37° C. for 1 hour. Phosphorylated bridging oligo 2 was then desalted using a Sephadex G-25 desalting column (Princeton Separations) and hybridized to the transcript and splint in a reaction containing 3.2 μM capped hEPO transcript, 1.5 μM bridging oligo 2 and 3 μM splint oligo 3 (or 1.5 uM splint oligo 7) by heating to 75° C. for 5 minutes followed by gradual cooling to room temperature over 5 minutes. An RNA ligation reaction was subsequently prepared to contain a 50% diluted hybridization reaction and 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.5 at 25° C.), 1 mM ATP, 10% PEG and 1 U/μL T4 RNA ligase 1 (NEB), and was reacted for 90 min at 37° C. The completed ligation reaction was then purified using an RNeasy Mini Kit (Qiagen).

EPO PCNA #2 (10A-10A Bridge): (SEQ ID NO: 18) 5′- GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAA GACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAAC GCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGGG UGCACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCU CCCUCUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGAC AGCCGAGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUA UCACGACGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGU CCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGG CAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUG UCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCC CCUGCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACC ACUCUGCUUCGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAG AUGCGGCCUCAGCUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCG CAAACUCUUCCGAGUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUG UACACAGGGGAGGCCUGCAGGACAGGGGACAGAUGACGGGUGGCAUCCC UGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAG UGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU AAAAAA AAAA -

- AAAAAAAAAA UCGAACUACGUUGAAUUAAAAUAAU CCUGUUCCGACCACCCGUGACCUCACCGUUGAAGGUCCCGGUCCUCUCC GUGACCCCUCCCCAGUGUCCCUACGGUGGGCAGUAGACAGGGGACAGGA CGUCCGGAGGGGACACAUGUCGAAGUCGAAAGGGGCCUCCUUUAACCUC AUCUGAGCCUUCUCAAACGCCUUUCACAGUCGUCACUAACAAGCCUCAC CUCGUCGACUCCGGCGUAGACCUCCCCUCUACCGAAGGAAGACCCGAGG GUCUCGGGCUUCGUCUCACCACUCCGACGCUUCCGGUGACUGCCGAAAU AGGUGUACGUCGACGUCCCCGAGGGUGCCGACCCUUCUCAACUGGUUGU CCCGGACCGGGGCGUCCUGUCGAAGGCUGUCGUCCCGGUCCGGGACGGU CUGAAGAUGCCGGACGACGGGCUGGAGGUAGGAGAAGGUCCGUAUCUUU AAUUGAAACCACAGACCCUGUCACUAUAAGAGUAAGUUCGACGUCACAA GUCGUGUCGGGCAGCACUAUAAGAGCCGGAGGAACCGGAGGUUCUCCAU GGAGAGGUCCUGAGCCGACAGUGUCUACUCCGCACCACCCCGCGGGUCC UGACCCUCCGGGUCUCCCUCGCUGUCGUCCCUGUCCUCUUCGGUGUCGG UCCGUCCUGUAAGCACGUGGGGGUAGCACAGUUCCUGCCACUCAGUGAG AACCGUGCCCCUUAGGCGCAAGGUUACGUGGCAAGGGCCGGCGCCUCCG ACCUAGCCAGGGCCACAGAAGAUACCUCCAGUUUUGUCGCACCUACCGC AGAGGUCCGCUAGACAGG-5′

EPO MCNA #3

MCNA 3 (SEQ ID NO: 19) was prepared by splint ligation of the 3′ end of two copies of an RNA encoding the human Erythropoietin (hEPO) protein to the 5′ ends of a single oligonucleotide containing two 5′ ends and a linked 3′-3′ phosphodiester bond within the sequence. Briefly, a 5′-capped RNA containing a 5′ untranslated region (UTR), a 3′ UTR with both UTRs flanking an RNA sequence encoding hEPO was transcribed using T7 RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure and purified. The construct was treated further to incorporate a poly(A) tail length of ˜200 As using poly(A) polymerase. This hEPO transcript was then ligated in a single step to OMeRNA “bridge” oligonucleotide containing a 20 nucleotide (nt) palindromic sequence with a 3′-3′ phosphodiester linkage between the 10^(th) and 11^(th) nt (bridging oligo 1 (SEQ ID NO: 5); 5′-CGA CUC UCG G-3′-3′-G GCU CUC AGC-5′, bold bases OMeRNA) using T4 RNA ligase 1+PEG 8K and a DNA oligonucleotide “splint” complementary to the 3′-UTR and bridging oligo 1 (splint oligo 4 (SEQ ID NO: 12); 5′ CCG AGA GTC GTT TTT TTT TTT TTT TTT TTT 3′; all bases DNA). Alternatively, MCNA could be prepared using splint oligo 8 (SEQ ID NO: 16), a palindromic sequence containing 2 copies of splint oligo 4 connected with a 5′-5′ phosphodiester bond. To prepare the samples for ligation, bridging oligo 1 was 5′-end phosphorylated in a reaction containing 50 μM oligo 1, 1 mM ATP, 1×PNK Buffer (NEB; 70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT, pH 7.6 at 25° C.) and 0.5 U/μL T4 Polynucleotide Kinase (NEB) at 37° C. for 1 hour. Phosphorylated bridging oligo 1 was then desalted using a Sephadex G-25 desalting column (Princeton Separations) and hybridized to the transcript and splint in a reaction containing 3.2 μM capped hEPO transcript, 1.5 μM bridging oligo 1 and 3 μM splint oligo 4 by heating to 75° C. for 5 minutes followed by gradual cooling to room temperature over 5 minutes. An RNA ligation reaction was subsequently prepared to contain a 50% diluted hybridization reaction and 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.5 at 25° C.), 1 mM ATP, 10% PEG and 1 U/μL T4 RNA ligase 1 (NEB), and was reacted for 90 minutes at 37° C. The completed ligation reaction was then purified using an RNeasy Mini Kit (Qiagen).

EPO PCNA #3 (includes 200A Poly(A) Tail): (SEQ ID NO: 19) 5′- GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAG ACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGC GGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGGGUGC ACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCU CUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGACAGCCG AGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUAUCACGA CGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGUCCCAGAC ACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGGCAGCAGGC CGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUGUCCUGCGGG GCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCUGCAGCUG CAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUUCG GGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAG CUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGA GUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGC CUGCAGGACAGGGGACAGAUGACGGGUGGCAUCCCUGUGACCCCUCCCCA GUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGU CCUAAUAAAAUUAAGUUGCAUCAAGCUAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA-

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA UCGAACUACGUUGAAUUAAAAUAAUCCUGUUCCGACCACCCGUGACCUCA CCGUUGAAGGUCCCGGUCCUCUCCGUGACCCCUCCCCAGUGUCCCUACGG UGGGCAGUAGACAGGGGACAGGACGUCCGGAGGGGACACAUGUCGAAGUC GAAAGGGGCCUCCUUUAACCUCAUCUGAGCCUUCUCAAACGCCUUUCACA GUCGUCACUAACAAGCCUCACCUCGUCGACUCCGGCGUAGACCUCCCCUC UACCGAAGGAAGACCCGAGGGUCUCGGGCUUCGUCUCACCACUCCGACGC UUCCGGUGACUGCCGAAAUAGGUGUACGUCGACGUCCCCGAGGGUGCCGA CCCUUCUCAACUGGUUGUCCCGGACCGGGGCGUCCUGUCGAAGGCUGUCG UCCCGGUCCGGGACGGUCUGAAGAUGCCGGACGACGGGCUGGAGGUAGGA GAAGGUCCGUAUCUUUAAUUGAAACCACAGACCCUGUCACUAUAAGAGUA AGUUCGACGUCACAAGUCGUGUCGGGCAGCACUAUAAGAGCCGGAGGAAC CGGAGGUUCUCCAUGGAGAGGUCCUGAGCCGACAGUGUCUACUCCGCACC ACCCCGCGGGUCCUGACCCUCCGGGUCUCCCUCGCUGUCGUCCCUGUCCU CUUCGGUGUCGGUCCGUCCUGUAAGCACGUGGGGGUAGCACAGUUCCUGC CACUCAGUGAGAACCGUGCCCCUUAGGCGCAAGGUUACGUGGCAAGGGCC GGCGCCUCCGACCUAGCCAGGGCCACAGAAGAUACCUCCAGUUUUGUCGC ACCUACCGCAGAGGUCCGCUAGACAGG-5′

EPO MCNA #4

MCNA 4 (SEQ ID NO: 20) was prepared by splint-independent ligation of the 3′-end of two copies of an RNA encoding the human Erythropoietin (hEPO) protein to the 5′-ends of a single dinucleotide containing two A's linked by a 3′-3′ phosphodiester bond. Briefly, a 5′-capped RNA containing a 5′-untranslated region (UTR), a 3′ UTR with both UTRs flanking an RNA sequence encoding hEPO was transcribed using T7 RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure and purified. The construct was treated further to incorporate a poly(A) tail length of ˜200 As using poly(A) polymerase. This hEPO transcript was then ligated via two steps to an RNA bridge oligonucleotide containing a trimeric repeat of As with a 3′-3′ phosphodiester linkage to another trimeric repeat of As (bridging oligo 3 (SEQ ID NO: 7); 5′-AAA-3′-3′-AAA-5′, underlined bases RNA) using T4 RNA ligase 1+PEG 8K. To prepare the samples for ligation, bridging oligo 3 was 5′-end phosphorylated in a reaction containing 50 μM oligo 7, 1 mM ATP, 1×PNK Buffer (NEB; 70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT, pH 7.6 at 25° C.) and 0.5 U/μL T4 Polynucleotide Kinase (NEB) at 37° C. for 1 hour. Phosphorylated bridging oligo 3 was then desalted using a Sephadex G-25 desalting column (Princeton Separations) and denatured in a reaction containing 2.4 μM capped and tailed hEPO transcript and 50 μM bridging oligo 3 by heating to 75° C. for 5 min followed by gradual cooling to room temperature over 5 min. An RNA ligation reaction was subsequently prepared to contain a 50% diluted hybridization reaction and 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT pH 7.5 at 25° C.), 1 mM ATP, 10% PEG and 1 U/μL T4 RNA ligase 1 (NEB), and was reacted for 90 minutes at 37° C. The partial ligation reaction was then purified using an RNeasy Mini Kit (Qiagen). The ligation reaction was repeated using a 1:1 molar ratio of the partial ligation product and additional capped and tailed hEPO transcript, and purified as previously.

EPO PCNA #4 (includes 200A Poly(A) Tail with 3A-3A Bridge): (SEQ ID NO: 20) 5′- GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAG ACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGC GGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGGGUGC ACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCU CUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGACAGCCG AGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUAUCACGA CGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGUCCCAGAC ACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGGCAGCAGGC CGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUGUCCUGCGGG GCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCUGCAGCUG CAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUUCG GGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAG CUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGA GUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGC CUGCAGGACAGGGGACAGAUGACGGGUGGCAUCCCUGUGACCCCUCCCCA GUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGU CCUAAUAAAAUUAAGUUGCAUCAAGCUAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA-

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA UCGAACUACGUUGAAUUAAAAUAAUCCUGUUCCGACCACCCGUGACCUCA CCGUUGAAGGUCCCGGUCCUCUCCGUGACCCCUCCCCAGUGUCCCUACGG UGGGCAGUAGACAGGGGACAGGACGUCCGGAGGGGACACAUGUCGAAGUC GAAAGGGGCCUCCUUUAACCUCAUCUGAGCCUUCUCAAACGCCUUUCACA GUCGUCACUAACAAGCCUCACCUCGUCGACUCCGGCGUAGACCUCCCCUC UACCGAAGGAAGACCCGAGGGUCUCGGGCUUCGUCUCACCACUCCGACGC UUCCGGUGACUGCCGAAAUAGGUGUACGUCGACGUCCCCGAGGGUGCCGA CCCUUCUCAACUGGUUGUCCCGGACCGGGGCGUCCUGUCGAAGGCUGUCG UCCCGGUCCGGGACGGUCUGAAGAUGCCGGACGACGGGCUGGAGGUAGGA GAAGGUCCGUAUCUUUAAUUGAAACCACAGACCCUGUCACUAUAAGAGUA AGUUCGACGUCACAAGUCGUGUCGGGCAGCACUAUAAGAGCCGGAGGAAC CGGAGGUUCUCCAUGGAGAGGUCCUGAGCCGACAGUGUCUACUCCGCACC ACCCCGCGGGUCCUGACCCUCCGGGUCUCCCUCGCUGUCGUCCCUGUCCU CUUCGGUGUCGGUCCGUCCUGUAAGCACGUGGGGGUAGCACAGUUCCUGC CACUCAGUGAGAACCGUGCCCCUUAGGCGCAAGGUUACGUGGCAAGGGCC GGCGCCUCCGACCUAGCCAGGGCCACAGAAGAUACCUCCAGUUUUGUCGC ACCUACCGCAGAGGUCCGCUAGACAGG-5′

EPO MCNA #5

MCNA 5 (SEQ ID NO: 21) was prepared by splint-independent ligation of the 3′ end of two copies of an RNA encoding the human Erythropoietin (hEPO) protein to the 5′ ends of a single dinucleotide containing two A's linked by a 3′-3′ phosphodiester bond. Briefly, a 5′-capped RNA containing a 5′-untranslated region (UTR), a 3′ UTR with both UTRs flanking an RNA sequence encoding hEPO was transcribed using T7 RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure and purified. The construct was treated further to incorporate a poly(A) tail length of ˜200 As using poly(A) polymerase. This hEPO transcript was then ligated via two steps to an RNA “bridging” dinucleotide containing an A with a 3′-3′ phosphodiester linkage to another A (bridging oligo 4 (SEQ ID NO: 8); 5′ A 3′ 3′ A 5′, underlined bases RNA) using T4 RNA ligase 1+PEG 8K. To prepare the samples for ligation, bridging oligo 4 was 5′-end phosphorylated in a reaction containing 50 μM bridging oligo 4, 1 mM ATP, 1×PNK Buffer (NEB; 70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT, pH 7.6 at 25° C.) and 0.5 U/μL T4 Polynucleotide Kinase (NEB) at 37° C. for 1 hour. Phosphorylated bridging oligo 4 was then desalted using a Sephadex G-25 desalting column (Princeton Separations) and denatured in a reaction containing 2.4 μM capped and tailed hEPO transcript and 50 μM bridging oligo 4 by heating to 75° C. for 5 minutes followed by gradual cooling to room temperature over 5 minutes. An RNA ligation reaction was subsequently prepared to contain a 50% diluted hybridization reaction and 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.5 at 25° C.), 1 mM ATP, 10% PEG and 1 U/μL T4 RNA ligase 1 (NEB), and was reacted for 90 minutes at 37° C. The partial ligation reaction was then purified using an RNeasy Mini Kit (Qiagen). The ligation reaction was repeated using a 1:1 molar ratio of the partial ligation product and additional capped and tailed hEPO transcript, and purified as previously.

EPO PCNA #5 (includes 200A Poly(A) Tail with 1A- 1A Bridge): (SEQ ID NO: 21) 5′- GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAG ACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGC GGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGGGUGC ACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCU CUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGACAGCCG AGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUAUCACGA CGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGUCCCAGAC ACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGGCAGCAGGC CGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUGUCCUGCGGG GCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCUGCAGCUG CAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUUCG GGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAG CUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGA GUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGC CUGCAGGACAGGGGACAGAUGACGGGUGGCAUCCCUGUGACCCCUCCCCA GUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGU CCUAAUAAAAUUAAGUUGCAUCAAGCUAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA-A-

′-A- AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA UCGAACUACGUUGAAUUAAAAUAAUCCUGUUCCGACCACCCGUGACCUCA CCGUUGAAGGUCCCGGUCCUCUCCGUGACCCCUCCCCAGUGUCCCUACGG UGGGCAGUAGACAGGGGACAGGACGUCCGGAGGGGACACAUGUCGAAGUC GAAAGGGGCCUCCUUUAACCUCAUCUGAGCCUUCUCAAACGCCUUUCACA GUCGUCACUAACAAGCCUCACCUCGUCGACUCCGGCGUAGACCUCCCCUC UACCGAAGGAAGACCCGAGGGUCUCGGGCUUCGUCUCACCACUCCGACGC UUCCGGUGACUGCCGAAAUAGGUGUACGUCGACGUCCCCGAGGGUGCCGA CCCUUCUCAACUGGUUGUCCCGGACCGGGGCGUCCUGUCGAAGGCUGUCG UCCCGGUCCGGGACGGUCUGAAGAUGCCGGACGACGGGCUGGAGGUAGGA GAAGGUCCGUAUCUUUAAUUGAAACCACAGACCCUGUCACUAUAAGAGUA AGUUCGACGUCACAAGUCGUGUCGGGCAGCACUAUAAGAGCCGGAGGAAC CGGAGGUUCUCCAUGGAGAGGUCCUGAGCCGACAGUGUCUACUCCGCACC ACCCCGCGGGUCCUGACCCUCCGGGUCUCCCUCGCUGUCGUCCCUGUCCU CUUCGGUGUCGGUCCGUCCUGUAAGCACGUGGGGGUAGCACAGUUCCUGC CACUCAGUGAGAACCGUGCCCCUUAGGCGCAAGGUUACGUGGCAAGGGCC GGCGCCUCCGACCUAGCCAGGGCCACAGAAGAUACCUCCAGUUUUGUCGC ACCUACCGCAGAGGUCCGCUAGACAGG-5′

EPO PCNA #6

PCNA 6 (SEQ ID NO: 22) is prepared by splint ligation of the 3′ end of two copies of an RNA encoding the human Erythropoietin (hEPO) protein to the 5′ ends of a single oligonucleotide containing two 5′ ends and a linked 3′-3′ phosphodiester bond within the sequence. Briefly, a 5′-capped RNA containing a 5′-untranslated region (UTR), a 3′ UTR containing an internal section of 65 consecutive As with both UTRs flanking an RNA sequence encoding hEPO is transcribed using T7 RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure and purified. This hEPO transcript is then ligated in a single step to a OMeRNA “bridging” oligonucleotide containing a 20 nucleotide (nt) palindromic sequence with a 3′-3′ phosphodiester linkage between the 10^(th) and 11^(th) nt (bridging oligo 1 (SEQ ID NO: 5); 5′-CGA CUC UCG G-3′-3′-G GCU CUC AGC-5′, underlined bases OMeRNA) using T4 RNA ligase 1+PEG 8K and a DNA oligonucleotide “splint” complementary to the 3′ UTR and bridging oligo 1 (splint oligo 1 (SEQ ID NO: 9); 5′ CCG AGA GTC GAG CTT GAT GCA ACT TAA TTT TAT TAG G 3′; all bases DNA). To prepare the samples for ligation, bridging oligo 1 is 5′-end phosphorylated in a reaction containing 50 μM bridging oligo 1, 1 mM ATP, 1×PNK Buffer (NEB; 70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT pH 7.6 at 25° C.) and 0.5 U/μL T4 Polynucleotide Kinase (NEB) at 37° C. for 1 hour. Phosphorylated bridging oligo 1 is then desalted using a Sephadex G-25 desalting column (Princeton Separations) and hybridized to the transcript and splint in a reaction containing 3.2 μM capped hEPO transcript, 1.5 μM bridging oligo 1 and 3 μM splint oligo 1 by heating to 75° C. for 5 minutes followed by gradual cooling to room temperature over 5 minutes. An RNA ligation reaction is subsequently prepared to contain a 50% diluted hybridization reaction and 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.5 at 25° C.), 1 mM ATP, 10% PEG and 1 U/μL T4 RNA ligase 1 (NEB), and is reacted for 90 minutes at 37° C. The completed ligation reaction is then purified using an RNeasy Mini Kit (Qiagen).

EPO PCNA #6 (includes internal 65A poly(A) region): (SEQ ID NO: 22) 5′- GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAA GACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAAC GCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGGG UGCACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCU CCCUCUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGAC AGCCGAGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUA UCACGACGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGU CCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGG CAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUG UCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCC CCUGCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACC ACUCUGCUUCGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAG AUGCGGCCUCAGCUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCG CAAACUCUUCCGAGUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUG UACACAGGGGAGGCCUGCAGGACAGGGGACAGAUGACGGGUGGCAUCCC UGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAG UGCCCACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAGCCUUGUCCUAAUAAAAUUAAGUUG CAUCAAGCU CGACUCUCGG-

GGCUCUCAGC UCGAACUACGUUGAAUUAAAAUAAUCCUGUUCCGAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA ACCACCCGUGACCUCACCGUUGAAGGUCCCGGUCCUCUCCGUGACCCCU CCCCAGUGUCCCUACGGUGGGCAGUAGACAGGGGACAGGACGUCCGGAG GGGACACAUGUCGAAGUCGAAAGGGGCCUCCUUUAACCUCAUCUGAGCC UUCUCAAACGCCUUUCACAGUCGUCACUAACAAGCCUCACCUCGUCGAC UCCGGCGUAGACCUCCCCUCUACCGAAGGAAGACCCGAGGGUCUCGGGC UUCGUCUCACCACUCCGACGCUUCCGGUGACUGCCGAAAUAGGUGUACG UCGACGUCCCCGAGGGUGCCGACCCUUCUCAACUGGUUGUCCCGGACCG GGGCGUCCUGUCGAAGGCUGUCGUCCCGGUCCGGGACGGUCUGAAGAUG CCGGACGACGGGCUGGAGGUAGGAGAAGGUCCGUAUCUUUAAUUGAAAC CACAGACCCUGUCACUAUAAGAGUAAGUUCGACGUCACAAGUCGUGUCG GGCAGCACUAUAAGAGCCGGAGGAACCGGAGGUUCUCCAUGGAGAGGUC CUGAGCCGACAGUGUCUACUCCGCACCACCCCGCGGGUCCUGACCCUCC GGGUCUCCCUCGCUGUCGUCCCUGUCCUCUUCGGUGUCGGUCCGUCCUG UAAGCACGUGGGGGUAGCACAGUUCCUGCCACUCAGUGAGAACCGUGCC CCUUAGGCGCAAGGUUACGUGGCAAGGGCCGGCGCCUCCGACCUAGCCA GGGCCACAGAAGAUACCUCCAGUUUUGUCGCACCUACCGCAGAGGUCCG CUAGACAGG-5′

EPO PCNA #7

PCNA7 (SEQ ID NO: 23) is prepared by splint ligation of the 3′ end of two copies of an RNA encoding the human Erythropoietin (hEPO) protein to the 5′ ends of a single oligonucleotide containing two 5′ ends and a linked 3′-3′ phosphodiester bond within the sequence. Briefly, a 5′-capped RNA containing a 5′ untranslated region (UTR), a 3′ UTR containing 3 stretches of 15 As and 1 stretch of 16 As with both UTRs flanking an RNA sequence encoding hEPO is transcribed using T7 RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure and purified. This hEPO transcript is then ligated in a single step to a OMeRNA “bridge” oligonucleotide containing a 20 nucleotide (nt) palindromic sequence with a 3′-3′ phosphodiester linkage between the 10^(th) and 11^(th) nt (bridging oligo 1 (SEQ ID NO: 5); 5′-CGA CUC UCG G-3′-3′-G GCU CUC AGC-5′, underlined bases OMeRNA) using T4 RNA ligase 1+PEG 8K and a DNA oligonucleotide “splint” complementary to the 3′-UTR and bridging oligo 1 (splint oligo 1 (SEQ ID NO: 9); 5′ CCG AGA GTC GAG CTT GAT GCA ACT TAA TTT TAT TAG G 3′; all bases DNA). To prepare the samples for ligation, oligo 1 is 5′-end phosphorylated in a reaction containing 50 μM bridging oligo 1, 1 mM ATP, 1×PNK Buffer (NEB; 70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT, pH 7.6 at 25° C.) and 0.5 U/μL T4 Polynucleotide Kinase (NEB) at 37° C. for 1 hour. Phosphorylated bridging oligo 1 is then desalted using a Sephadex G-25 desalting column (Princeton Separations) and hybridized to the transcript and splint in a reaction containing 3.2 μM capped hEPO transcript, 1.5 μM bridging oligo 1 and 3 μM splint oligo 1 by heating to 75° C. for 5 minutes followed by gradual cooling to room temperature over 5 minutes. An RNA ligation reaction is subsequently prepared to contain a 50% diluted hybridization reaction and 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.5 at 25° C.), 1 mM ATP, 10% PEG and 1 U/μL T4 RNA ligase 1 (NEB), and is reacted for 90 min at 37° C. The completed ligation reaction is then purified using an RNeasy Mini Kit (Qiagen).

EPO PCNA #7 (includes multiple short internal poly(A) regions): (SEQ ID NO: 23) 5′- GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGA AGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGA ACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGG GGGUGCACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGU CGCUCCCUCUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCU GUGACAGCCGAGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCG AGAAUAUCACGACGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUA UCACUGUCCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGG AGGUCGGGCAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGU CGGAAGCUGUCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGC CGUGGGAGCCCCUGCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUC GCAGCCUCACCACUCUGCUUCGGGCUCUGGGAGCCCAGAAGGAAGCCA UCUCCCCUCCAGAUGCGGCCUCAGCUGCUCCACUCCGAACAAUCACUG CUGACACUUUCCGCAAACUCUUCCGAGUCUACUCCAAUUUCCUCCGGG GAAAGCUGAAGCUGUACACAGGGGAGGCCUGCAGGACAGGGGACAGAU GACGGGUGGCAAAAAAAAAAAAAAAUCCCUGUGACCCCUCCCCAAAAA AAAAAAAAAAAGUGCCUCUCCUGGCCCUGGAAAAAAAAAAAAAAAGUU GCCACUCCAGUGCCCACCAAAAAAAAAAAAAAAGCCUUGUCCUAAUAA AAUUAAGUUGCAUCAAGCU CGACUCUCGG-

GGCUCUCA GC UCGAACUACGUUGAAUUAAAAUAAUCCUGUUCCGAAAAAAAAAAAA AAACCACCCGUGACCUCACCGUUGAAAAAAAAAAAAAAAGGUCCCGGU CCUCUCCGUGAAAAAAAAAAAAAAAACCCCUCCCCAGUGUCCCUAAAA AAAAAAAAAAACGGUGGGCAGUAGACAGGGGACAGGACGUCCGGAGGG GACACAUGUCGAAGUCGAAAGGGGCCUCCUUUAACCUCAUCUGAGCCU UCUCAAACGCCUUUCACAGUCGUCACUAACAAGCCUCACCUCGUCGAC UCCGGCGUAGACCUCCCCUCUACCGAAGGAAGACCCGAGGGUCUCGGG CUUCGUCUCACCACUCCGACGCUUCCGGUGACUGCCGAAAUAGGUGUA CGUCGACGUCCCCGAGGGUGCCGACCCUUCUCAACUGGUUGUCCCGGA CCGGGGCGUCCUGUCGAAGGCUGUCGUCCCGGUCCGGGACGGUCUGAA GAUGCCGGACGACGGGCUGGAGGUAGGAGAAGGUCCGUAUCUUUAAUU GAAACCACAGACCCUGUCACUAUAAGAGUAAGUUCGACGUCACAAGUC GUGUCGGGCAGCACUAUAAGAGCCGGAGGAACCGGAGGUUCUCCAUGG AGAGGUCCUGAGCCGACAGUGUCUACUCCGCACCACCCCGCGGGUCCU GACCCUCCGGGUCUCCCUCGCUGUCGUCCCUGUCCUCUUCGGUGUCGG UCCGUCCUGUAAGCACGUGGGGGUAGCACAGUUCCUGCCACUCAGUGA GAACCGUGCCCCUUAGGCGCAAGGUUACGUGGCAAGGGCCGGCGCCUC CGACCUAGCCAGGGCCACAGAAGAUACCUCCAGUUUUGUCGCACCUAC CGCAGAGGUCCGCUAGACAGG-5′

FIG. 5 shows the results of MCNA detected via gel electrophoresis. MCNA run in lanes 1-15 were the result of a ligation reaction comprising an EPO mRNA to bridging oligonucleotide to DNA splint (SEQ ID NO: 9) molar ratio of 2:1:2. The molar amounts of EPO mRNA and RNA ligase are included in the below table:

Lane EPO (μM) Ligase (μM) 1 1.7 2.25 RNA Ligase 1 2 1.7 0.6 RNA Ligase 1 3 0.85 0.6 RNA Ligase 1 4 0.425 0.6 RNA Ligase 1 5 0.2125 0.6 RNA Ligase 1 6 1.7 2.25 RNA Ligase 1 + 10% PEG 7 1.7 0.6 RNA Ligase 1 + 10% PEG 8 0.85 0.6 RNA Ligase 1 + 10% PEG 9 0.425 0.6 RNA Ligase 1 + 10% PEG 10 0.2125 0.6 RNA Ligase 1 + 10% PEG 11 1.7 0.3 RNA Ligase 2 12 1.7 0.6 RNA Ligase 2 13 0.85 0.6 RNA Ligase 2 14 0.425 0.6 RNA Ligase 2 15 0.2125 0.6 RNA Ligase 2 FIG. 5 demonstrates that RNA Ligase 1 was superior to RNA Ligase 2 in producing MCNA comprising EPO RNA under the conditions tested. Further, the addition of 10% PEG to the reaction conditions enhanced ligation.

FIG. 6 shows MCNA detected via gel electrophoresis. Lane 1 shows Capped EPO mRNA (no poly(A) tail). Lane 2 shows a MCNA mixture of full length MCNA ligation product mixed with unreacted/partially reacted EPO RNA product (with no DNAse treatment). Lane 3 shows a MCNA mixture of full length MCNA ligation product mixed with unreacted/partially reacted EPO RNA product (with DNAse treatment).

FIG. 8 shows MCNA detected via gel electrophoresis. Lane 1 shows a RNA sizing ladder. Lane 2 shows a MCNA mixture of full length MCNA ligation product mixed with unreacted/partially reacted EPO RNA product. Lane 3 shows purified unreacted/partially reacted EPO RNA product. Lane 4 shows purified EPO MCNA ligation product.

MCNA-OTC Preparation

MCNA-OTC comprising human Ornithine Transcarbamylase (hOTC) RNA (SEQ ID NO: 24) was prepared by splint ligation of the 3′-end of two copies of an RNA encoding the hOTC protein to the 5′-ends of a single oligonucleotide containing two 5′ ends and a linked 3′-3′ phosphodiester bond within the sequence. Briefly, a 5′-capped RNA containing a 5′-untranslated region (UTR) and a 3′ UTR flanking an RNA sequence encoding hOTC was transcribed using RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure and purified. This hOTC transcript was then ligated in a single step to a 2′-hydroxymethylated RNA (OMeRNA) “bridge” oligonucleotide containing a 20 nucleotide (nt) palindromic sequence with a 3′-3′ phosphodiester linkage between the 10^(th) and 11^(th) nt (oligo 1 (bridge) (SEQ ID NO: 5); 5′-CGA CUC UCG G-3′-3′-G GCU CUC AGC-5′, bold bases OMeRNA) using either T4 RNA ligase 1+PEG 8K and a DNA oligonucleotide “splint” complementary to the 3′-UTR and oligo 1 (oligo 2 (splint) (SEQ ID NO: 9); 5′ CCG AGA GTC GAG CTT GAT GCA ACT TAA TTT TAT TAG G 3′; all bases DNA). To prepare the samples for ligation, oligo 1 was 5′-end phosphorylated in a reaction containing 50 μM oligo 1, 1 mM ATP, 1×PNK Buffer (NEB; 70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT pH 7.6 at 25° C.) and 0.5 U/μL T4 Polynucleotide Kinase (NEB) at 37° C. for 1 hour. Phosphorylated oligo 1 (bridge) was then desalted using a Sephadex G-25 desalting column (Princeton Separations) and hybridized to the transcript and splint in a reaction containing 3.3 μM capped hOTC transcript, 1.5 μM oligo 1 and 3.3 μM oligo 2 by heating to 75° C. for 5 minutes, followed by gradual cooling to room temperature over 5 minutes. An RNA ligation reaction was subsequently prepared to contain a 50% diluted hybridization reaction and 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT pH 7.5 at 25° C.), 1 mM ATP, 10% PEG and 0.33 U/μL T4 RNA ligase 1. Each was reacted for 60 minutes at 37° C. The completed ligation reaction was then reacted with DNase I and subsequently purified using an RNeasy Maxi Kit (Qiagen). The reaction products were evaluated for ligation efficiency using TBE/agarose gel electrophoresis. The isolated MCNA-OTC product was equilibrated with Lipofectamine and transfected into adherent HEK293 cells. Unfractionated cell lysate was then assayed for citrulline production from ornithine and carbamoyl phosphate (FIG. 10).

MCNA-OTC (SEQ ID NO: 24) 5′- GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAA GACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAAC GCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGCUGU UCAACCUUCGGAUCUUGCUGAACAACGCUGCGUUCCGGAAUGGUCACAA CUUCAUGGUCCGGAACUUCAGAUGCGGCCAGCCGCUCCAGAACAAGGUG CAGCUCAAGGGGAGGGACCUCCUCACCCUGAAAAACUUCACCGGAGAAG AGAUCAAGUACAUGCUGUGGCUGUCAGCCGACCUCAAAUUCCGGAUCAA GCAGAAGGGCGAAUACCUUCCUUUGCUGCAGGGAAAGUCCCUGGGGAUG AUCUUCGAGAAGCGCAGCACUCGCACUAGACUGUCAACUGAAACCGGCU UCGCGCUGCUGGGAGGACACCCCUGCUUCCUGACCACCCAAGAUAUCCA UCUGGGUGUGAACGAAUCCCUCACCGACACAGCGCGGGUGCUGUCGUCC AUGGCAGACGCGGUCCUCGCCCGCGUGUACAAGCAGUCUGAUCUGGACA CUCUGGCCAAGGAAGCCUCCAUUCCUAUCAUUAAUGGAUUGUCCGACCU CUACCAUCCCAUCCAGAUUCUGGCCGAUUAUCUGACUCUGCAAGAACAU UACAGCUCCCUGAAGGGGCUUACCCUUUCGUGGAUCGGCGACGGCAACA ACAUUCUGCACAGCAUUAUGAUGAGCGCUGCCAAGUUUGGAAUGCACCU CCAAGCAGCGACCCCGAAGGGAUACGAGCCAGACGCCUCCGUGACGAAG CUGGCUGAGCAGUACGCCAAGGAGAACGGCACUAAGCUGCUGCUCACCA ACGACCCUCUCGAAGCCGCCCACGGUGGCAACGUGCUGAUCACCGAUAC CUGGAUCUCCAUGGGACAGGAGGAGGAAAAGAAGAAGCGCCUGCAAGCA UUUCAGGGGUACCAGGUGACUAUGAAAACCGCCAAGGUCGCCGCCUCGG ACUGGACCUUCUUGCACUGUCUGCCCAGAAAGCCCGAAGAGGUGGACGA CGAGGUGUUCUACAGCCCGCGGUCGCUGGUCUUUCCGGAGGCCGAAAAC AGGAAGUGGACUAUCAUGGCCGUGAUGGUGUCCCUGCUGACCGAUUACU CCCCGCAGCUGCAGAAACCAAAGUUCUGACGGGUGGCAUCCCUGUGACC CCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCAC CAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU

UCGAACUACGUUGAAUUAAAAUAAUCCUGUUCCGACCA CCCGUGACCUCACCGUUGAAGGUCCCGGUCCUCUCCGUGACCCCUCCCC AGUGUCCCUACGGUGGGCAGUCUUGAAACCAAAGACGUCGACGCCCCUC AUUAGCCAGUCGUCCCUGUGGUAGUGCCGGUACUAUCAGGUGAAGGACA AAAGCCGGAGGCCUUUCUGGUCGCUGGCGCCCGACAUCUUGUGGAGCAG CAGGUGGAGAAGCCCGAAAGACCCGUCUGUCACGUUCUUCCAGGUCAGG CUCCGCCGCUGGAACCGCCAAAAGUAUCAGUGGACCAUGGGGACUUUAC GAACGUCCGCGAAGAAGAAAAGGAGGAGGACAGGGUACCUCUAGGUCCA UAGCCACUAGUCGUGCAACGGUGGCACCCGCCGAAGCUCUCCCAGCAAC CACUCGUCGUCGAAUCACGGCAAGAGGAACCGCAUGACGAGUCGGUCGA AGCAGUGCCUCCGCAGACCGAGCAUAGGGAAGCCCCAGCGACGAACCUC CACGUAAGGUUUGAACCGUCGCGAGUAGUAUUACGACACGUCUUACAAC AACGGCAGCGGCUAGGUGCUUUCCCAUUCGGGGAAGUCCCUCGACAUUA CAAGAACGUCUCAGUCUAUUAGCCGGUCUUAGACCUACCCUACCAUCUC CAGCCUGUUAGGUAAUUACUAUCCUUACCUCCGAAGGAACCGGUCUCAC AGGUCUAGUCUGACGAACAUGUGCGCCCGCUCCUGGCGCAGACGGUACC UGCUGUCGUGGGCGCGACACAGCCACUCCCUAAGCAAGUGUGGGUCUAC CUAUAGAACCCACCAGUCCUUCGUCCCCACAGGAGGGUCGUCGCGCUUC GGCCAAAGUCAACUGUCAGAUCACGCUCACGACGCGAAGAGCUUCUAGU AGGGGUCCCUGAAAGGGACGUCGUUUCCUUCCAUAAGCGGGAAGACGAA CUAGGCCUUAAACUCCAGCCGACUGUCGGUGUCGUACAUGAACUAGAGA AGAGGCCACUUCAAAAAGUCCCACUCCUCCAGGGAGGGGAACUCGACGU GGAACAAGACCUCGCCGACCGGCGUAGACUUCAAGGCCUGGUACUUCAA CACUGGUAAGGCCUUGCGUCGCAACAAGUCGUUCUAGGCUUCCAACUUG UCGUAGCACAGUUCCUGCCACUCAGUGAGAACCGUGCCCCUUAGGCGCA AGGUUACGUGGCAAGGGCCGGCGCCUCCGACCUAGCCAGGGCCACAGAA GAUACCUCCAGUUUUGUCGCACCUACCGCAGAGGUCCGCUAGACAGG- 5′ (Bold base are OMeRNA)  MCNA-PAH Preparation

MCNA-PAH comprising human Phenylalanine Hydroxylase (hPAH) RNA (SEQ ID NO: 25) was prepared by splint ligation of the 3′-end of two copies of an RNA encoding the hPAH protein to the 5′-ends of a single oligonucleotide containing two 5′ ends and a linked 3′-3′ phosphodiester bond within the sequence. Briefly, a 5′-capped RNA containing a 5′-untranslated region (UTR) and a 3′ UTR flanking an RNA sequence encoding hPAH was transcribed using RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure and purified. This hPAH transcript was then ligated in a single step to a 2′-hydroxymethylated RNA (OMeRNA) “bridge” oligonucleotide containing a 20 nucleotide (nt) palindromic sequence with a 3′-3′ phosphodiester linkage between the 10^(th) and 11^(th) nt (oligo 1 (bridge) (SEQ ID NO: 5); 5′-CGA CUC UCG G-3′-3′-G GCU CUC AGC-5′, bold bases OMeRNA) using either T4 RNA ligase 1+PEG 8K and a DNA oligonucleotide “splint” complementary to the 3′-UTR and oligo 1 (oligo 2 (splint) (SEQ ID NO: 9); 5′ CCG AGA GTC GAG CTT GAT GCA ACT TAA TTT TAT TAG G 3′; all bases DNA). To prepare the samples for ligation, oligo 1 was 5′-end phosphorylated in a reaction containing 50 μM oligo 1, 1 mM ATP, 1×PNK Buffer (NEB; 70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT pH 7.6 at 25° C.) and 0.5 U/μL T4 Polynucleotide Kinase (NEB) at 37° C. for 1 hour. Phosphorylated oligo 1 (bridge) was then desalted using a Sephadex G-25 desalting column (Princeton Separations) and hybridized to the transcript and splint in a reaction containing 2.7 μM capped hPAH transcript, 1.2 μM oligo 1 and 2.7 μM oligo 2 by heating to 75° C. for 5 minutes, followed by gradual cooling to room temperature over 5 minutes. An RNA ligation reaction was subsequently prepared to contain a 50% diluted hybridization reaction and 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT pH 7.5 at 25° C.), 1 mM ATP, 10% PEG and 0.33 U/μL T4 RNA ligase 1. Each was reacted for 60 minutes at 37° C. The completed ligation reaction was then reacted with DNase I and subsequently purified using an RNeasy Maxi Kit (Qiagen). The reaction products were evaluated for ligation efficiency using TBE/agarose gel electrophoresis. The isolated MCNA-PAH reaction product was equilibrated with Lipofectamine and transfected into adherent HEK293 cells. Unfractionated cell lysate was then assayed for PAH protein expression using a PAH-specific ELISA (FIG. 11).

MCNA-PAH (SEQ ID NO: 25) 5′- GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAA GACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAAC GCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGAGCA CCGCCGUGCUGGAGAACCCCGGCCUGGGCCGCAAGCUGAGCGACUUCGG CCAGGAGACCAGCUACAUCGAGGACAACUGCAACCAGAACGGCGCCAUC AGCCUGAUCUUCAGCCUGAAGGAGGAGGUGGGCGCCCUGGCCAAGGUGC UGCGCCUGUUCGAGGAGAACGACGUGAACCUGACCCACAUCGAGAGCCG CCCCAGCCGCCUGAAGAAGGACGAGUACGAGUUCUUCACCCACCUGGAC AAGCGCAGCCUGCCCGCCCUGACCAACAUCAUCAAGAUCCUGCGCCACG ACAUCGGCGCCACCGUGCACGAGCUGAGCCGCGACAAGAAGAAGGACAC CGUGCCCUGGUUCCCCCGCACCAUCCAGGAGCUGGACCGCUUCGCCAAC CAGAUCCUGAGCUACGGCGCCGAGCUGGACGCCGACCACCCCGGCUUCA AGGACCCCGUGUACCGCGCCCGCCGCAAGCAGUUCGCCGACAUCGCCUA CAACUACCGCCACGGCCAGCCCAUCCCCCGCGUGGAGUACAUGGAGGAG GAGAAGAAGACCUGGGGCACCGUGUUCAAGACCCUGAAGAGCCUGUACA AGACCCACGCCUGCUACGAGUACAACCACAUCUUCCCCCUGCUGGAGAA GUACUGCGGCUUCCACGAGGACAACAUCCCCCAGCUGGAGGACGUGAGC CAGUUCCUGCAGACCUGCACCGGCUUCCGCCUGCGCCCCGUGGCCGGCC UGCUGAGCAGCCGCGACUUCCUGGGCGGCCUGGCCUUCCGCGUGUUCCA CUGCACCCAGUACAUCCGCCACGGCAGCAAGCCCAUGUACACCCCCGAG CCCGACAUCUGCCACGAGCUGCUGGGCCACGUGCCCCUGUUCAGCGACC GCAGCUUCGCCCAGUUCAGCCAGGAGAUCGGCCUGGCCAGCCUGGGCGC CCCCGACGAGUACAUCGAGAAGCUGGCCACCAUCUACUGGUUCACCGUG GAGUUCGGCCUGUGCAAGCAGGGCGACAGCAUCAAGGCCUACGGCGCCG GCCUGCUGAGCAGCUUCGGCGAGCUGCAGUACUGCCUGAGCGAGAAGCC CAAGCUGCUGCCCCUGGAGCUGGAGAAGACCGCCAUCCAGAACUACACC GUGACCGAGUUCCAGCCCCUGUACUACGUGGCCGAGAGCUUCAACGACG CCAAGGAGAAGGUGCGCAACUUCGCCGCCACCAUCCCCCGCCCCUUCAG CGUGCGCUACGACCCCUACACCCAGCGCAUCGAGGUGCUGGACAACACC CAGCAGCUGAAGAUCCUGGCCGACAGCAUCAACAGCGAGAUCGGCAUCC UGUGCAGCGCCCUGCAGAAGAUCAAGUAACGGGUGGCAUCCCUGUGACC CCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCAC CAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU

UCGAACUACGUUGAAU UAAAAUAAUCCUGUUCCGACCACCCGUGACCUCACCGUUGAAGGUCCCG GUCCUCUCCGUGACCCCUCCCCAGUGUCCCUACGGUGGGCAAUGAACUA GAAGACGUCCCGCGACGUGUCCUACGGCUAGAGCGACAACUACGACAGC CGGUCCUAGAAGUCGACGACCCACAACAGGUCGUGGAGCUACGCGACCC ACAUCCCCAGCAUCGCGUGCGACUUCCCCGCCCCCUACCACCGCCGCUU CAACGCGUGGAAGAGGAACCGCAGCAACUUCGAGAGCCGGUGCAUCAUG UCCCCGACCUUGAGCCAGUGCCACAUCAAGACCUACCGCCAGAAGAGGU CGAGGUCCCCGUCGUCGAACCCGAAGAGCGAGUCCGUCAUGACGUCGAG CGGCUUCGACGAGUCGUCCGGCCGCGGCAUCCGGAACUACGACAGCGGG ACGAACGUGUCCGGCUUGAGGUGCCACUUGGUCAUCUACCACCGGUCGA AGAGCUACAUGAGCAGCCCCCGCGGGUCCGACCGGUCCGGCUAGAGGAC CGACUUGACCCGCUUCGACGCCAGCGACUUGUCCCCGUGCACCGGGUCG UCGAGCACCGUCUACAGCCCGAGCCCCCACAUGUACCCGAACGACGGCA CCGCCUACAUGACCCACGUCACCUUGUGCGCCUUCCGGUCCGGCGGGUC CUUCAGCGCCGACGAGUCGUCCGGCCGGUGCCCCGCGUCCGCCUUCGGC CACGUCCAGACGUCCUUGACCGAGUGCAGGAGGUCGACCCCCUACAACA GGAGCACCUUCGGCGUCAUGAAGAGGUCGUCCCCCUUCUACACCAACAU GAGCAUCGUCCGCACCCAGAACAUGUCCGAGAAGUCCCAGAACUUGUGC CACGGGGUCCAGAAGAAGAGGAGGAGGUACAUGAGGUGCGCCCCCUACC CGACCGGCACCGCCAUCAACAUCCGCUACAGCCGCUUGACGAACGCCGC CCGCGCCAUGUGCCCCAGGAACUUCGGCCCCACCAGCCGCAGGUCGAGC CGCGGCAUCGAGUCCUAGACCAACCGCUUCGCCAGGUCGAGGACCUACC ACGCCCCCUUGGUCCCGUGCCACAGGAAGAAGAACAGCGCCGAGUCGAG CACGUGCCACCGCGGCUACAGCACCGCGUCCUAGAACUACUACAACCAG UCCCGCCCGUCCGACGCGAACAGGUCCACCCACUUCUUGAGCAUGAGCA GGAAGAAGUCCGCCGACCCCGCCGAGAGCUACACCCAGUCCAAGUGCAG CAAGAGGAGCUUGUCCGCGUCGUGGAACCGGUCCCGCGGGUGGAGGAGG AAGUCCGACUUCUAGUCCGACUACCGCGGCAAGACCAACGUCAACAGGA GCUACAUCGACCAGAGGACCGGCUUCAGCGAGUCGAACGCCGGGUCCGG CCCCAAGAGGUCGUGCCGCCACGAGUAGCACAGUUCCUGCCACUCAGUG AGAACCGUGCCCCUUAGGCGCAAGGUUACGUGGCAAGGGCCGGCGCCUC CGACCUAGCCAGGGCCACAGAAGAUACCUCCAGUUUUGUCGCACCUACC GCAGAGGUCCGCUAGACAGG-5′ (Bold base are OMeRNA) MCNA-CFTR Preparation

MCNA-CFTR comprising human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR) RNA (SEQ ID NO: 26) was prepared by splint ligation of the 3′-end of two copies of an RNA encoding the hCFTR protein to the 5′-ends of a single oligonucleotide containing two 5′ ends and a linked 3′-3′ phosphodiester bond within the sequence. Briefly, a 5′-capped RNA containing a 5′-untranslated region (UTR) and a 3′ UTR flanking an RNA sequence encoding hCFTR was transcribed using RNA polymerase, enzymatically capped to contain a 5′-Cap 1 structure and purified. This hCFTR transcript was then ligated in a single step to a 2′-hydroxymethylated RNA (OMeRNA) “bridge” oligonucleotide containing a 20 nucleotide (nt) palindromic sequence with a 3′-3′ phosphodiester linkage between the 10^(th) and 11^(th) nt (oligo 1 (bridge) (SEQ ID NO: 5); 5′-CGA CUC UCG G-3′-3′-G GCU CUC AGC-5′, bold bases OMeRNA) using either T4 RNA ligase 1+PEG 8K and a DNA oligonucleotide “splint” complementary to the 3′-UTR and oligo 1 (oligo 2 (splint) (SEQ ID NO: 9); 5′ CCG AGA GTC GAG CTT GAT GCA ACT TAA TTT TAT TAG G 3′; all bases DNA). To prepare the samples for ligation, oligo 1 was 5′-end phosphorylated in a reaction containing 50 μM oligo 1, 1 mM ATP, 1×PNK Buffer (NEB; 70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT pH 7.6 at 25° C.) and 0.5 U/μL T4 Polynucleotide Kinase (NEB) at 37° C. for 1 hour. Phosphorylated oligo 1 (bridge) was then desalted using a Sephadex G-25 desalting column (Princeton Separations) and hybridized to the transcript and splint in a reaction containing 0.92 μM capped hCFTR transcript, 0.42 μM oligo 1 and 0.92 μM oligo 2 by heating to 75° C. for 5 minutes followed by gradual cooling to room temperature over 5 minutes. An RNA ligation reaction was subsequently prepared to contain a 50% diluted hybridization reaction and 1×RNA ligase Buffer (NEB; 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT pH 7.5 at 25° C.), 1 mM ATP, 10% PEG and 0.33 U/μL T4 RNA ligase 1. Each was reacted for 60 minutes at 37° C. The completed ligation reaction was then reacted with DNase I and subsequently purified using an RNeasy Maxi Kit (Qiagen). The reaction products were evaluated for ligation efficiency using TBE/agarose gel electrophoresis. The isolated MCNA-CFTR product was equilibrated with Lipofectamine and transfected into adherent HEK293 cells. Unfractionated cell lysate was then assayed for CFTR protein expression using CFTR-specific Western Blotting (FIG. 12).

MCNA-CFTR (SEQ ID NO: 26) 5′- GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAA GACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAAC GCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGCAAC GCUCUCCUCUUGAAAAGGCCUCGGUGGUGUCCAAGCUCUUCUUCUCGUG GACUAGACCCAUCCUGAGAAAGGGGUACAGACAGCGCUUGGAGCUGUCC GAUAUCUAUCAAAUCCCUUCCGUGGACUCCGCGGACAACCUGUCCGAGA AGCUCGAGAGAGAAUGGGACAGAGAACUCGCCUCAAAGAAGAACCCGAA GCUGAUUAAUGCGCUUAGGCGGUGCUUUUUCUGGCGGUUCAUGUUCUAC GGCAUCUUCCUCUACCUGGGAGAGGUCACCAAGGCCGUGCAGCCCCUGU UGCUGGGACGGAUUAUUGCCUCCUACGACCCCGACAACAAGGAAGAAAG AAGCAUCGCUAUCUACUUGGGCAUCGGUCUGUGCCUGCUUUUCAUCGUC CGGACCCUCUUGUUGCAUCCUGCUAUUUUCGGCCUGCAUCACAUUGGCA UGCAGAUGAGAAUUGCCAUGUUUUCCCUGAUCUACAAGAAAACUCUGAA GCUCUCGAGCCGCGUGCUUGACAAGAUUUCCAUCGGCCAGCUCGUGUCC CUGCUCUCCAACAAUCUGAACAAGUUCGACGAGGGCCUCGCCCUGGCCC ACUUCGUGUGGAUCGCCCCUCUGCAAGUGGCGCUUCUGAUGGGCCUGAU CUGGGAGCUGCUGCAAGCCUCGGCAUUCUGUGGGCUUGGAUUCCUGAUC GUGCUGGCACUGUUCCAGGCCGGACUGGGGCGGAUGAUGAUGAAGUACA GGGACCAGAGAGCCGGAAAGAUUUCCGAACGGCUGGUGAUCACUUCGGA AAUGAUCGAAAACAUCCAGUCAGUGAAGGCCUACUGCUGGGAAGAGGCC AUGGAAAAGAUGAUUGAAAACCUCCGGCAAACCGAGCUGAAGCUGACCC GCAAGGCCGCUUACGUGCGCUAUUUCAACUCGUCCGCUUUCUUCUUCUC CGGGUUCUUCGUGGUGUUUCUCUCCGUGCUCCCCUACGCCCUGAUUAAG GGAAUCAUCCUCAGGAAGAUCUUCACCACCAUUUCCUUCUGUAUCGUGC UCCGCAUGGCCGUGACCCGGCAGUUCCCAUGGGCCGUGCAGACUUGGUA CGACUCCCUGGGAGCCAUUAACAAGAUCCAGGACUUCCUUCAAAAGCAG GAGUACAAGACCCUCGAGUACAACCUGACUACUACCGAGGUCGUGAUGG AAAACGUCACCGCCUUUUGGGAGGAGGGAUUUGGCGAACUGUUCGAGAA GGCCAAGCAGAACAACAACAACCGCAAGACCUCGAACGGUGACGACUCC CUCUUCUUUUCAAACUUCAGCCUGCUCGGGACGCCCGUGCUGAAGGACA UUAACUUCAAGAUCGAAAGAGGACAGCUCCUGGCGGUGGCCGGAUCGAC CGGAGCCGGAAAGACUUCCCUGCUGAUGGUGAUCAUGGGAGAGCUUGAA CCUAGCGAGGGAAAGAUCAAGCACUCCGGCCGCAUCAGCUUCUGUAGCC AGUUUUCCUGGAUCAUGCCCGGAACCAUUAAGGAAAACAUCAUCUUCGG CGUGUCCUACGAUGAAUACCGCUACCGGUCCGUGAUCAAAGCCUGCCAG CUGGAAGAGGAUAUUUCAAAGUUCGCGGAGAAAGAUAACAUCGUGCUGG GCGAAGGGGGUAUUACCUUGUCGGGGGGCCAGCGGGCUAGAAUCUCGCU GGCCAGAGCCGUGUAUAAGGACGCCGACCUGUAUCUCCUGGACUCCCCC UUCGGAUACCUGGACGUCCUGACCGAAAAGGAGAUCUUCGAAUCGUGCG UGUGCAAGCUGAUGGCUAACAAGACUCGCAUCCUCGUGACCUCCAAAAU GGAGCACCUGAAGAAGGCAGACAAGAUUCUGAUUCUGCAUGAGGGGUCC UCCUACUUUUACGGCACCUUCUCGGAGUUGCAGAACUUGCAGCCCGACU UCUCAUCGAAGCUGAUGGGUUGCGACAGCUUCGACCAGUUCUCCGCCGA AAGAAGGAACUCGAUCCUGACGGAAACCUUGCACCGCUUCUCUUUGGAA GGCGACGCCCCUGUGUCAUGGACCGAGACUAAGAAGCAGAGCUUCAAGC AGACCGGGGAAUUCGGCGAAAAGAGGAAGAACAGCAUCUUGAACCCCAU UAACUCCAUCCGCAAGUUCUCAAUCGUGCAAAAGACGCCACUGCAGAUG AACGGCAUUGAGGAGGACUCCGACGAACCCCUUGAGAGGCGCCUGUCCC UGGUGCCGGACAGCGAGCAGGGAGAAGCCAUCCUGCCUCGGAUUUCCGU GAUCUCCACUGGUCCGACGCUCCAAGCCCGGCGGCGGCAGUCCGUGCUG AACCUGAUGACCCACAGCGUGAACCAGGGCCAAAACAUUCACCGCAAGA CUACCGCAUCCACCCGGAAAGUGUCCCUGGCACCUCAAGCGAAUCUUAC CGAGCUCGACAUCUACUCCCGGAGACUGUCGCAGGAAACCGGGCUCGAA AUUUCCGAAGAAAUCAACGAGGAGGAUCUGAAAGAGUGCUUCUUCGACG AUAUGGAGUCGAUACCCGCCGUGACGACUUGGAACACUUAUCUGCGGUA CAUCACUGUGCACAAGUCAUUGAUCUUCGUGCUGAUUUGGUGCCUGGUG AUUUUCCUGGCCGAGGUCGCGGCCUCACUGGUGGUGCUCUGGCUGUUGG GAAACACGCCUCUGCAAGACAAGGGAAACUCCACGCACUCGAGAAACAA CAGCUAUGCCGUGAUUAUCACUUCCACCUCCUCUUAUUACGUGUUCUAC AUCUACGUCGGAGUGGCGGAUACCCUGCUCGCGAUGGGUUUCUUCAGAG GACUGCCGCUGGUCCACACCUUGAUCACCGUCAGCAAGAUUCUUCACCA CAAGAUGUUGCAUAGCGUGCUGCAGGCCCCCAUGUCCACCCUCAACACU CUGAAGGCCGGAGGCAUUCUGAACAGAUUCUCCAAGGACAUCGCUAUCC UGGACGAUCUCCUGCCGCUUACCAUCUUUGACUUCAUCCAGCUGCUGCU GAUCGUGAUUGGAGCAAUCGCAGUGGUGGCGGUGCUGCAGCCUUACAUU UUCGUGGCCACUGUGCCGGUCAUUGUGGCGUUCAUCAUGCUGCGGGCCU ACUUCCUCCAAACCAGCCAGCAGCUGAAGCAACUGGAAUCCGAGGGACG AUCCCCCAUCUUCACUCACCUUGUGACGUCGUUGAAGGGACUGUGGACC CUCCGGGCUUUCGGACGGCAGCCCUACUUCGAAACCCUCUUCCACAAGG CCCUGAACCUCCACACCGCCAAUUGGUUCCUGUACCUGUCCACCCUGCG GUGGUUCCAGAUGCGCAUCGAGAUGAUUUUCGUCAUCUUCUUCAUCGCG GUCACAUUCAUCAGCAUCCUGACUACCGGAGAGGGAGAGGGACGGGUCG GAAUAAUCCUGACCCUCGCCAUGAACAUUAUGAGCACCCUGCAGUGGGC AGUGAACAGCUCGAUCGACGUGGACAGCCUGAUGCGAAGCGUCAGCCGC GUGUUCAAGUUCAUCGACAUGCCUACUGAGGGAAAACCCACUAAGUCCA CUAAGCCCUACAAAAAUGGCCAGCUGAGCAAGGUCAUGAUCAUCGAAAA CUCCCACGUGAAGAAGGACGAUAUUUGGCCCUCCGGAGGUCAAAUGACC GUGAAGGACCUGACCGCAAAGUACACCGAGGGAGGAAACGCCAUUCUCG AAAACAUCAGCUUCUCCAUUUCGCCGGGACAGCGGGUCGGCCUUCUCGG GCGGACCGGUUCCGGGAAGUCAACUCUGCUGUCGGCUUUCCUCCGGCUG CUGAAUACCGAGGGGGAAAUCCAAAUUGACGGCGUGUCUUGGGAUUCCA UUACUCUGCAGCAGUGGCGGAAGGCCUUCGGCGUGAUCCCCCAGAAGGU GUUCAUCUUCUCGGGUACCUUCCGGAAGAACCUGGAUCCUUACGAGCAG UGGAGCGACCAAGAAAUCUGGAAGGUCGCCGACGAGGUCGGCCUGCGCU CCGUGAUUGAACAAUUUCCUGGAAAGCUGGACUUCGUGCUCGUCGACGG GGGAUGUGUCCUGUCGCACGGACAUAAGCAGCUCAUGUGCCUCGCACGG UCCGUGCUCUCCAAGGCCAAGAUUCUGCUGCUGGACGAACCUUCGGCCC ACCUGGAUCCGGUCACCUACCAGAUCAUCAGGAGGACCCUGAAGCAGGC CUUUGCCGAUUGCACCGUGAUUCUCUGCGAGCACCGCAUCGAGGCCAUG CUGGAGUGCCAGCAGUUCCUGGUCAUCGAGGAGAACAAGGUCCGCCAAU ACGACUCCAUUCAAAAGCUCCUCAACGAGCGGUCGCUGUUCAGACAAGC UAUUUCACCGUCCGAUAGAGUGAAGCUCUUCCCGCAUCGGAACAGCUCA AAGUGCAAAUCGAAGCCGCAGAUCGCAGCCUUGAAGGAAGAGACUGAGG AAGAGGUGCAGGACACCCGGCUUUAACGGGUGGCAUCCCUGUGACCCCU CCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAG CCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU

UCGAACUACGUUGAAUUA AAAUAAUCCUGUUCCGACCACCCGUGACCUCACCGUUGAAGGUCCCGGU CCUCUCCGUGACCCCUCCCCAGUGUCCCUACGGUGGGCAAUUUCGGCCC ACAGGACGUGGAGAAGGAGUCAGAGAAGGAAGUUCCGACGCUAGACGCC GAAGCUAAACGUGAAACUCGACAAGGCUACGCCCUUCUCGAAGUGAGAU AGCCUGCCACUUUAUCGAACAGACUUGUCGCUGGCGAGCAACUCCUCGA AAACUUACCUCAGCAUAACCGCCUGGAACAAGAGGAGCUACUGGUCCUU GACGACCGUGAGGUCGUACCGGAGCUACGCCACGAGCGUCUCUUAGUGC CACGUUAGCCGUUUCCGGACGAAGUCCCAGGAGGACUACUAGACCAUCC ACUGGCCUAGGUCCACCCGGCUUCCAAGCAGGUCGUCGUCUUAGAACCG GAACCUCUCGUGCCUGGCACGCUCCGUGUACUCGACGAAUACAGGCACG CUGUCCUGUGUAGGGGGCAGCUGCUCGUGCUUCAGGUCGAAAGGUCCUU UAACAAGUUAGUGCCUCGCGUCCGGCUGGAGCAGCCGCUGGAAGGUCUA AAGAACCAGCGAGGUGACGAGCAUUCCUAGGUCCAAGAAGGCCUUCCAU GGGCUCUUCUACUUGUGGAAGACCCCCUAGUGCGGCUUCCGGAAGGCGG UGACGACGUCUCAUUACCUUAGGGUUCUGUGCGGCAGUUAAACCUAAAG GGGGAGCCAUAAGUCGUCGGCCUCCUUUCGGCUGUCGUCUCAACUGAAG GGCCUUGGCCAGGCGGGCUCUUCCGGCUGGGCGACAGGGCCGCUUUACC UCUUCGACUACAAAAGCUCUUACCGCAAAGGAGGGAGCCACAUGAAACG CCAGUCCAGGAAGUGCCAGUAAACUGGAGGCCUCCCGGUUUAUAGCAGG AAGAAGUGCACCCUCAAAAGCUACUAGUACUGGAACGAGUCGACCGGUA AAAACAUCCCGAAUCACCUGAAUCACCCAAAAGGGAGUCAUCCGUACAG CUACUUGAACUUGUGCGCCGACUGCGAAGCGUAGUCCGACAGGUGCAGC UAGCUCGACAAGUGACGGGUGACGUCCCACGAGUAUUACAAGUACCGCU CCCAGUCCUAAUAAGGCUGGGCAGGGAGAGGGAGAGGCCAUCAGUCCUA CGACUACUUACACUGGCGCUACUUCUUCUACUGCUUUUAGUAGAGCUAC GCGUAGACCUUGGUGGCGUCCCACCUGUCCAUGUCCUUGGUUAACCGCC ACACCUCCAAGUCCCGGAACACCUUCUCCCAAAGCUUCAUCCCGACGGC AGGCUUUCGGGCCUCCCAGGUGUCAGGGAAGUUGCUGCAGUGUUCCACU CACUUCUACCCCCUAGCAGGGAGCCUAAGGUCAACGAAGUCGACGACCG ACCAAACCUCCUUCAUCCGGGCGUCGUACUACUUGCGGUGUUACUGGCC GUGUCACCGGUGCUUUUACAUUCCGACGUCGUGGCGGUGGUGACGCUAA CGAGGUUAGUGCUAGUCGUCGUCGACCUACUUCAGUUUCUACCAUUCGC CGUCCUCUAGCAGGUCCUAUCGCUACAGGAACCUCUUAGACAAGUCUUA CGGAGGCCGGAAGUCUCACAACUCCCACCUGUACCCCCGGACGUCGUGC GAUACGUUGUAGAACACCACUUCUUAGAACGACUGCCACUAGUUCCACA CCUGGUCGCCGUCAGGAGACUUCUUUGGGUAGCGCUCGUCCCAUAGGCG GUGAGGCUGCAUCUACAUCUUGUGCAUUAUUCUCCUCCACCUUCACUAU UAGUGCCGUAUCGACAACAAAGAGCUCACGCACCUCAAAGGGAACAGAA CGUCUCCGCACAAAGGGUUGUCGGUCUCGUGGUGGUCACUCCGGCGCUG GAGCCGGUCCUUUUAGUGGUCCGUGGUUUAGUCGUGCUUCUAGUUACUG AACACGUGUCACUACAUGGCGUCUAUUCACAAGGUUCAGCAGUGCCGCC CAUAGCUGAGGUAUAGCAGCUUCUUCGUGAGAAAGUCUAGGAGGAGCAA CUAAAGAAGCCUUUAAAGCUCGGGCCAAAGGACGCUGUCAGAGGCCCUC AUCUACAGCUCGAGCCAUUCUAAGCGAACUCCACGGUCCCUGUGAAAGG CCCACCUACGCCAUCAGAACGCCACUUACAAAACCGGGACCAAGUGCGA CACCCAGUAGUCCAAGUCGUGCCUGACGGCGGCGGCCCGAACCUCGCAG CCUGGUCACCUCUAGUGCCUUUAGGCUCCGUCCUACCGAAGAGGGACGA GCGACAGGCCGUGGUCCCUGUCCGCGGAGAGUUCCCCAAGCAGCCUCAG GAGGAGUUACGGCAAGUAGACGUCACCGCAGAAAACGUGCUAACUCUUG AACGCCUACCUCAAUUACCCCAAGUUCUACGACAAGAAGGAGAAAAGCG GCUUAAGGGGCCAGACGAACUUCGAGACGAAGAAUCAGAGCCAGGUACU GUGUCCCCGCAGCGGAAGGUUUCUCUUCGCCACGUUCCAAAGGCAGUCC UAGCUCAAGGAAGAAAGCCGCCUCUUGACCAGCUUCGACAGCGUUGGGU AGUCGAAGCUACUCUUCAGCCCGACGUUCAAGACGUUGAGGCUCUUCCA CGGCAUUUUCAUCCUCCUGGGGAGUACGUCUUAGUCUUAGAACAGACGG AAGAAGUCCACGAGGUAAAACCUCCAGUGCUCCUACGCUCAGAACAAUC GGUAGUCGAACGUGUGCGUGCUAAGCUUCUAGAGGAAAAGCCAGUCCUG CAGGUCCAUAGGCUUCCCCCUCAGGUCCUCUAUGUCCAGCCGCAGGAAU AUGUGCCGAGACCGGUCGCUCUAAGAUCGGGCGACCGGGGGGCUGUUCC AUUAUGGGGGAAGCGGGUCGUGCUACAAUAGAAAGAGGCGCUUGAAACU UUAUAGGAGAAGGUCGACCGUCCGAAACUAGUGCCUGGCCAUCGCCAUA AGUAGCAUCCUGUGCGGCUUCUACUACAAAAGGAAUUACCAAGGCCCGU ACUAGGUCCUUUUGACCGAUGUCUUCGACUACGCCGGCCUCACGAACUA GAAAGGGAGCGAUCCAAGUUCGAGAGGGUACUAGUGGUAGUCGUCCCUU CAGAAAGGCCGAGGCCAGCUAGGCCGGUGGCGGUCCUCGACAGGAGAAA GCUAGAACUUCAAUUACAGGAAGUCGUGCCCGCAGGGCUCGUCCGACUU CAAACUUUUCUUCUCCCUCAGCAGUGGCAAGCUCCAGAACGCCAACAAC AACAAGACGAACCGGAAGAGCUUGUCAAGCGGUUUAGGGAGGAGGGUUU UCCGCCACUGCAAAAGGUAGUGCUGGAGCCAUCAUCAGUCCAACAUGAG CUCCCAGAACAUGAGGACGAAAACUUCCUUCAGGACCUAGAACAAUUAC CGAGGGUCCCUCAGCAUGGUUCAGACGUGCCGGGUACCCUUGACGGCCC AGUGCCGGUACGCCUCGUGCUAUGUCUUCCUUUACCACCACUUCUAGAA GGACUCCUACUAAGGGAAUUAGUCCCGCAUCCCCUCGUGCCUCUCUUUG UGGUGCUUCUUGGGCCUCUUCUUCUUUCGCCUGCUCAACUUUAUCGCGU GCAUUCGCCGGAACGCCCAGUCGAAGUCGAGCCAAACGGCCUCCAAAAG UUAGUAGAAAAGGUACCGGAGAAGGGUCGUCAUCCGGAAGUGACUGACC UACAAAAGCUAGUAAAGGCUUCACUAGUGGUCGGCAAGCCUUUAGAAAG GCCGAGAGACCAGGGACAUGAAGUAGUAGUAGGCGGGGUCAGGCCGGAC CUUGUCACGGUCGUGCUAGUCCUUAGGUUCGGGUGUCUUACGGCUCCGA ACGUCGUCGAGGGUCUAGUCCGGGUAGUCUUCGCGGUGAACGUCUCCCC GCUAGGUGUGCUUCACCCGGUCCCGCUCCGGGAGCAGCUUGAACAAGUC UAACAACCUCUCGUCCCUGUGCUCGACCGGCUACCUUUAGAACAGUUCG UGCGCCGAGCUCUCGAAGUCUCAAAAGAACAUCUAGUCCCUUUUGUACC GUUAAGAGUAGACGUACGGUUACACUACGUCCGGCUUUUAUCGUCCUAC GUUGUUCUCCCAGGCCUGCUACUUUUCGUCCGUGUCUGGCUACGGGUUC AUCUAUCGCUACGAAGAAAGAAGGAACAACAGCCCCAGCAUCCUCCGUU AUUAGGCAGGGUCGUUGUCCCCGACGUGCCGGAACCACUGGAGAGGGUC CAUCUCCUUCUACGGCAUCUUGUACUUGGCGGUCUUUUUCGUGGCGGAU UCGCGUAAUUAGUCGAAGCCCAAGAAGAAACUCCGCUCAAGAGACAGGG UAAGAGAGAGCUCGAAGAGCCUGUCCAACAGGCGCCUCAGGUGCCUUCC CUAAACUAUCUAUAGCCUGUCGAGGUUCGCGACAGACAUGGGGAAAGAG UCCUACCCAGAUCAGGUGCUCUUCUUCUCGAACCUGUGGUGGCUCCGGA AAAGUUCUCCUCUCGCAACGUAGCACAGUUCCUGCCACUCAGUGAGAAC CGUGCCCCUUAGGCGCAAGGUUACGUGGCAAGGGCCGGCGCCUCCGACC UAGCCAGGGCCACAGAAGAUACCUCCAGUUUUGUCGCACCUACCGCAGA GGUCCGCUAGACAGG-5′ (Bold base are OMeRNA)

Example 2. Exemplary Protein Production with MCNA

This example demonstrates the production of protein encoded by mRNA linked by their 3′ ends to a bridging oligonucleotide.

MCNA comprising human erythropoietin (hEPO) mRNA were synthesized as described above and used to transfect HEK293T cells (1 microgram RNA transfection per sample). FIG. 7 shows the results of an experiment comparing the amount of secreted hEPO protein from HEK293T cells when the cells were transfected with either a) mRNA encoding hEPO that lacked a polyA tail, b) MCNA comprising hEPO mRNA, or c) MCNA comprising hEPO mRNA that had been treated with DNase. A clear increase in protein production was achieved when the cells were transfected with either the MCNA comprising hEPO mRNA or the DNase-treated MCNA comprising hEPO mRNA compared to the untailed hEPO mRNA.

FIG. 9 shows the results of an experiment comparing the amount of secreted hEPO protein from HEK293T cells when the cells were transfected with either a) mRNA encoding hEPO that lacked a polyA tail, b) unpurified mixture of MCNA comprising hEPO mRNA with unreacted/partially reacted EPO RNA, c) purified unreacted/partially reacted EPO RNA, or d) purified EPO MCNA. All samples were transfected with a total of 250 nanograms RNA. A clear increase in protein production was achieved when the cells were transfected with purified EPO MCNA compared to the mixture or unreacted hEPO RNA. FIG. 10 shows the results of an experiment comparing the amount of human OTC protein activity (as measured by citrulline production) within HEK293T cells when the cells were transfected with either a) mRNA encoding hOTC that lacked a polyA tail (hOTC monomer), or b) MCNA comprising hOTC mRNA. Detectable protein production was achieved only when the cells were transfected with the MCNA comprising hOTC as compared to the hOTC monomer.

FIG. 11 shows the results of an experiment comparing the amount of human PAH protein produced within HEK293T cells when the cells were transfected with either a) mRNA encoding hPAH that lacked a polyA tail (hPAH monomer), or b) MCNA comprising hPAH mRNA. Significantly higher protein production was achieved when the cells were transfected with the MCNA comprising hPAH as compared to the hPAH monomer.

FIG. 12 shows the results of an experiment comparing the amount of human CFTR protein produced within HEK293T cells when the cells were transfected with either a) mRNA encoding hCFTR that lacked a polyA tail (hCFTR monomer), or b) MCNA comprising hCFTR mRNA. Detectable protein production was achieved only when the cells were transfected with the MCNA comprising hCFTR as compared to the hCFTR monomer.

Example 3. Exemplary In Vivo Protein Production with MCNA

This example demonstrates the in vivo production of protein encoded by mRNA linked by their 3′ ends to a bridging oligonucleotide.

MCNA comprising human ornithine carbamoyltransferase (hOTC) mRNA were synthesized as described above. spf^(ash) mice were treated intravenously with hOTC MCNA encapsulated in lipid nanoparticles. Animals were sacrificed and their livers were isolated either 24 hours or 7 days post-administration. Citrulline production was measured in the liver samples and it was found that the level of hOTC protein activity 7 days post-administration was comparable to the level of hOTC protein activity 24 hours post-administration (FIG. 13). At both time points, hOTC protein activity was significantly greater than in the livers of control spf^(ash) mice. Further, substantial hOTC protein was detected via Western blot at both 1 day and 8 days post-administration, but for only the spf^(ash) mice treated with hOTC MCNA LNPs, not the mice treated with the hOTC monomer LNPs (FIG. 14), consistent with the observed activity data. In comparison, when spf^(ash) mice were treated intravenously with hOTC mRNA, levels of hOTC protein activity were higher 24 hours post-administration than they were 7 days post-administration (FIG. 15). As clearly shown in FIG. 16, when hOTC protein activity 7 day post-administration was calculated as a percentage of activity levels after 24 hours, more sustained in vivo activity is observed for hOTC MCNA (109% of 24 hour activity) than for hOTC mRNA (38% of 24 hour activity).

In another study, MCNA comprising human phenylalanine hydroxylase (hPAH) were synthesized as described above. PAH knock-out (KO) mice were treated intravenously with either hPAH MCNA or an hPAH monomer (hPAH mRNA with a 5′ cap but without a polyA tail) encapsulated in lipid nanoparticles. Animals were sacrificed and their livers were isolated 24 hours post-administration. More than 27 times more hPAH protein was detected in the livers of mice treated with hPAH MCNA than was detected in the livers of mice treated with the hPAH monomer (FIG. 17).

Further, a demonstration of efficacy was achieved after treatment of PAH knock-out (KO) mice with hPAH MCNA LNPs. Specifically, serum phenylalanine levels were significantly reduced 24 hours after treatment with hPAH MCNA while no reduction in serum phenylalanine was seen 24 hours after treatment with hPAH monomer LNPs (FIG. 18).

In another study, MCNA comprising human erythropoietin (hEPO) were synthesized as described above. Wild-type mice were treated intravenously with either hEPO MCNA or an hEPO monomer (hEPO mRNA with a 5′ cap but without a polyA tail) encapsulated in lipid nanoparticles. Serum samples from the animals were obtained 24 hours post-administration. More than 480 times more hEPO protein was detected in the serum of mice treated with hEPO MCNA than was detected in the serum of mice treated with the hEPO monomer (FIG. 19).

In another study, MCNA comprising human cystic fibrosis transmembrane conductance regulator (hCFTR) were synthesized as described above. CFTR KO mice were treated via aerosolization of hCFTR MCNA encapsulated in lipid nanoparticles. Animals were sacrificed and their lungs were isolated either 24 hours or 7 days post-administration. As shown in FIG. 20, MCNA-derived hCFTR protein was detected in both the bronchial epithelial airways (top row) as well as alveolar regions (bottom row) both 24 hours and 7 days post-administration (brown staining).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims: 

We claim:
 1. A method of delivering a multimeric coding nucleic acid (MCNA) for in vivo protein production, comprising administering to a subject in need of the MCNA, wherein the MCNA comprises two messenger RNAs (mRNAs) linked at 3′ ends, via a stable linkage, such that the multimeric coding nucleic acid has two 5′ ends, and wherein the stable linkage is an oligonucleotide bridge comprising an internal 3′-to-3′ inverted phosphodiester linkage.
 2. The method of claim 1, wherein each of the mRNAs encodes a protein of interest.
 3. The method of claim 2, wherein each of the mRNAs encodes a same protein.
 4. The method of claim 2, wherein each of the mRNAs encodes a distinct protein.
 5. The method of claim 1, wherein the mRNAs comprise a 3′ UTR.
 6. The method of claim 5, wherein the 3′ UTR comprises a plurality of multi-A segments with spacers in between.
 7. The method of claim 1, wherein the oligonucleotide bridge comprises nucleosides selected from the group consisting of 2′-OMe-A, 2′-OMe-G, 2′-OMe-C, 2′-OMe-U, 2′-F-A, 2′-F-G, 2′-F-C, 2′-F-U, LNA-A, LNA-G, LNA-C, LNA-U, N6-methyl-adenosine, 2-thiouridine (2sU), 5-methyl-cytidine (5mC), pseudouridine (ΨU), and 1-methyl-pseudouridine.
 8. The method of claim 1, wherein the mRNAs comprise one or more modified nucleosides.
 9. The method of claim 8, wherein the modified nucleosides are selected from the group consisting of 2′-OMe-A, 2′-OMe-G, 2′-OMe-C, 2′-OMe-U, 2′-F-A, 2′-F-G, 2′-F-C, 2′-F-U, LNA-A, LNA-G, LNA-C, LNA-U, N6-methyl-adenosine, 2-thiouridine (2sU), 5-methyl-cytidine (5mC), pseudouridine (ΨU), and 1-methyl-pseudouridine.
 10. The method of claim 1, wherein each of the mRNAs encodes an enzyme, a receptor, a ligand, a light chain or heavy chain of an antibody, a nuclease, or a DNA-binding protein.
 11. The method of claim 1, wherein the MCNA is encapsulated or complexed with a delivery vehicle.
 12. The method of claim 11, wherein the delivery vehicle is selected from the group consisting of liposomes, lipid nanoparticles, solid-lipid nanoparticles, polymers, viruses, sol-gels, and nanogels.
 13. The method of claim 5, wherein the 3′ UTR does not include a polyA tail.
 14. The method of claim 1, wherein each of the mRNAs is unmodified.
 15. The method of claim 1, wherein the oligonucleotide bridge is unmodified.
 16. The method of claim 1, wherein each of the mRNAs has a 5′ cap. 